Ettropm~ .hcrmrl ufPltartnncology, 204 ( 199 I )55-h I 99 WI Elsevier Science Publishers B.V. All rights reserved t~l4-~999/9~

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ADEINIS 00142YY9910070,‘U

EJP 51?101

Received 31 January 1991, revised MS received 14 June 1991, accepted 6 August 1991

The protein kinasc C stimulator phorbol 12-myristatc-1Iacetate (PMA) increased the release of noradrenaline from fieId-stimulated mouse vasa deferentia and antagonized the inhibitor effect of xylazine and FK 33-824. The mechanical response to field stimulation was not modified by PMA in unpretreated vasa, but the contractility was partly restored when the stimulation-response curve had been depressed by xylazine or FK 33-824. In contrast, PMA decreased the contractile response to exogenous noradrenaline and b~thanechol. A similar but smaller reduction was obtained with 1,2-dioctauoyi-sn-glycerol. In vasa depolarized by KCI, PMA and verapamil reduced the amplitude of contractions elicited by CaCI,. Only the effect of vcrapamil could bc reversed by the calcium ionophore A 23187. while the effect of PMA was greatly attenuated in muscles pretreated with ouabain. Phorbol 12-myristate-l3-acetate-4-~-mcthyiether was ineffective in all experiments. These results suggest that PMA increases noradrenaline release (prcjunctional cffcct) and dccreascs vasal contractility (postjunctional effect) by activation of protein kinase C. Phorbol ester: Protein kinase C; Field stimulation:

Noradrcnafine

1. lntmhction The Ca’+- and phospholipid-dependent protein kinase C (PKC) is present in most mammalian neuronal and non-neoronal tissues (Yoshida et al., 1988). The enzyme phospho~lates a variety of proteins including, for instance, ion channel (Shearman et al., 1989) and contractile proteins, such as troponins I and T (Katoh et al., 1983). and myosin light chains (Chatterjee and Tejada, 1986). The functional significance of these effects is not always clear. The natural activator of PKC, dia~ylglycerol, is produced by enzymatic cleavage of phosphatidylinositol 4J-biphosphate. Inositol phospholipid tu~over is increased by stimulation of many receptors (Watson and Godfrey, 1988), including muscarinic cholinoceptors and cr,-adrenoceptors; thus activation of PKC may be involved in the generation of receptor-mediated effects. Diacylglycerol cannot be used in in vitro experiments because it does not readily cross the cell membrane, but certain phorbol esters seem to be appropriate substitutes (Kaczmarek, 1987).

Corresp~)ndene~ to: i-1. Brasch. lnstitut fiir Pi~ar~la~~~~~ie. Medizinische Lbivcrsitiit zu Liibeck. Ratzeburger Allee 160. D-Z400 Liiheck 1. F.R.G.

release; Contractility; Vas deferens (mouse)

These drugs enhance the release of neurotransmitters from brain slices (Allgaier et al., 1987, 19881, isolated heart preparations (Ishac and De Luca, 1988; Musgrave and Majewski, 1989) and rat salivary glands (Wakade et al., 1985). Their influence on muscle contractions is less uniform. Phorbol esters produce a negative inotropic effect in the heart (Yuan et al., 1987; Teutsch et al., 1987) but induce long-lasting contractions in rabbit iris fHowe et al., 19861, guinea pig lung parenchymal strips (Dole and Obianime, 1985) and vascular smooth muscle (Forder et al., 1985; itoh and Lederis, 1988). The tonic contraction of guinea pig ileum is inhibited while the initial phasic contraction is augmented (Sasaguri and Watson. 1989). Finally, both contracting (Park and Rasmussen, 1986) and relaxing (Menkes et al., 1986) effects of phorbol esters have been described in isolated trachea preparations. Thus. the influence of PKC activation on the contractile force seems to vary considerably in the different types of smooth muscle. Little is known about the influence of PKC stimulation on the vas deferens. Baraban et al. (1985) have reported that phorbol esters enhance noradrenaline-induced contractions in the rat vas, but as far as we know the present paper is the first systematic investigation of the pre- and postjunctionel effects of phorbol lo-myristate-13-acetate (PMA) in a vas deferens preparation.

fr{~m their mesenteric sheaths and mounted in a S-ml c~rg;m bath containing modified Krebs solution of the f~~l~~~\v~~g composition (mmol/l): NaCl 11X; KC1 3.X; CaCl, 2.54: NaHCQ, 25; KH .PO, 0.93; glucose I I and Ca-ED-I-A 0.1 I. The solution was kept at 32 ’ C and gassed with carbogcn (9% 0, + 5’-; CO,). The preload of the vas was adjusted to I mN and contractions \vere registered isometrically via a K 30 forcedisplacement transducer (Hugo Sachs. Hugstctten. FRG) on a Servogor S recorder (Goerz Elektronik. Wien. Austria). In field stimulation experiments. the vasa were stimulated electrically with square pulses (28 V; approximatdy 200 mA) applied via a pair of platinum ring electrodes. During a l-h equilibration period. l-s trains of pulses ((1.1 ms: 15 Hz) were applied every 30 s. Thereafter up to four consecutive stimulation-response curves were obtained from each vas at IS-min internals.. One second trains of pulses were applied every 30 s and tht- intensity was increased by stcpwise increases in the pulse width (0.04. 0.06. 0.1, 0.1, 0.3 and 0.5 ms) at a constant (15 Hz) stimulation frequency. The first stimulation-response curve served as control while test drugs were added 1S min before the second to fourth curves. Non-cumulative concentration-response curves for noradrenaline or bethanechol were constructed when the vasa had equilibrated in the bathing solution for at least 1 h. Increasing concentrations of either noradrenaline (0.6-240 PM) or bethanechol (20-400 /IM) were then added to the organ bath and could act on the vas for 30 s each. The maxima of the resulting phasic contractions were evaluated. The application intervals were 5 min for noradrenaline and 10 min for bethanechol. Three consecutive concentration-response curves were obtained for each vas. The first one served as control while phorbol esters or dioctanoylglycerol was added 15 min before the second and third curves. To establish concentration-response curves for catcium. vasa were incubated in a depolarizing solution which contained 43 mmol/l NaCl and 80 mmol/l KCI, no CaC12 but 0.11 mmol/l Ca-EDTA. Three consecutive non-cumulative concentration-response curves were constructed. Increasing concentrations of CaCI, (0.5-d mM) were added to the organ bath at 5-min intervals and stayed in contact with the vas for 30 s each. An interval of 15 min was allowed between two successive curves and drugs were added 1j min before the second and third curves.

For the noradrenaline release experiments, both vasa of one mouse were tied in parallel and incubated for 1 h with 10 PCi DL-7-[‘Hlnoradrenaline. They were then carefully rinsed with Krcbs solution. transferred to a fresh organ bath and mounted under a preload of 5 mN. During the next hour the bathing solution was changed every 10 min. Cocaine (3 PM) was added after the end of the washing period and was present during the subsequent release experiments to prevent neuronal reuptake of noradrenaline. During the release experiment, 1 ml samples of the bathing fluid were taken every 2.5 min and the bathing fluid was renewed immediately after sample taking. Noradrenaline release was stimulated during the 3rd, 7th and I lth sampling period (stimulation periods S,, S, and S,) by applying 10 I-s trains of electrical field pulses ( 15 Hz frequency 0.5 ms pulse duration) at 15-s intervals. At the end of the experiments, the vasa were soluhilized in I ml Soluene 350 (Packard Instruments, Frankfurt, FRG) for determination of residual radioactivity.

2.2 C&rlatiom

and statistics

The field stimulation-induced release of radioactivity from the vasa was taken as a reliable indicator of the release of noradrenaline (Marshall, 1983; Stjirne and Astrand, 1985). The release of radioactivity was measured by mixing the l-ml samples of the bathing fluid with 9 ml scintillation fluid (Hydroluma”; Baker, Deventer, Netherlands) and counting for 10 min in a scintillation counter (BF 5003; Laboratorium Dr. Berthold, Wildbad, FRG). The stimulation-induced release of radioactivity during the three stimulation periods (S,. SZ and S,) was calculated by subtracting basal unstimulated release determined from the 2nd, 6th or 10th collection period from the total release measured for the 3rd, 7th or 1lth collection period. The stimulation-induced release was then expressed as fractional release, i.e. the quotient of radioactivity released riuring the stimulation period and total tissue content of radioactivity at the beginning of the stimulation period. For noradrenaline and bethanechol, ECs,, values and their 95% confidence limits were calculated from the log concentration-response curves after linearization by logit transformation and estimation of the regression function by the least-squares method. In the release experiments, !%/S, and S,/S, ratios for the fractional release of radioactivity were calculatel1. Arithmetic means and their S.E.s (n = 5-6) are pr::sented in the figures. Student’s t-test for paired data was used to evaluate drug effects on the fractional release and a probability level of P < 0.05 (two-tailed) was chosen as threshold of statistical significance.

57

2.3. Drugs The following drugs were used in the study: DL-7hydrochloride (Amersham Buchler. Braunschweig, FRG; specific activity lo-20 pCi/mmol); cocaine hydrochloride (Merck, Darmstadt, FRG); bethanechol hydrochloride; phorbol 12myristate-13-acetate; phorbol 12-myristate-13-acetateIt-0-methylether; [D-Ala”,Me,Phe’,Met(0)-ol-s]enkephalin (FK 33-824); ouabain; ionophore A 23187; 1,2-dioctanoyl-sn-glycerol (all Sigma, Deizenhofen. FRG); noradrenaline-I-hydrogentartrate (Fluka, Buchs, Switzerland); ryanodine (Calbiochem. Frankfurt, FRG) and verapamil hydrochloride (Knoll, Ludwigshafen, FRG). The phorbols, ionophore A 23187 and dioctanolglycerol were dissolved in dimethyl sulfoxide; all other drugs were dissolved in double distilled water. Stock solutions were prepared daily and microlitre amounts were added to the organ bath. [ ‘Hlnoradrenaline

3. Results 3. I. Field sti~nulatim~

Field stimulation with l-s trains of pulses (15 Hz) produced twitch contractions of the vasa. The force of contractioq increased with increasing pulse duration. For the control curves, the maximum developed force ranged between 12.9 f 0.8 and 17.7 +_1.7 mN in the different groups of muscles (fig. 1). In control experiments, four almost identical stimulation-response curves could be produced in the same vasa (not shown). In concentrations up to 30 PM, neither PMA (fig. IA) nor phorbol 12-myristate-13-acetate-4-0-methylether (PME; not shown) had a significant influence on the stimulation-induced twitch contractions.

The a,-adrenoceptor agonist xylazine (1 PM) depressed the stimulation-response curve and reduced the maximum developed force by about 35-504. This effect was partly reversed by 10 and 30 PM PMA (fig. IB) while 30 PM PME was ineffective in this respect (not shown). The p-receptor agonist FK 33-824 (1.36 PM) also depressed the stimulation-response curve and the maximum developed force by about 38-45%. Again this effect could be reversed by PMA (fig. 10 while no significant reduction was observed with PME (not shown).

3.2. Noradrenaline

release

There was a continuous efflux of radioactivity from vasa preloaded with [‘Hlnoradrenaline, even when they were not stimulated electrically. ‘H-Labelled noradrenaline as well as its various metabolites may have contributed to this basal efflux. which was not modified by any of the drugs under study. Field stimulation induced an additional release of radioactivity, which probably reflects the release of noradrenaline from sympathetic nerve terminals (see Methods). During the first stimulation period (S,), about l-2% of the to?al content of radioactivity in the vasa was released. In control experiments, this fractional release was quite similar during the second and third stimulation periods and thus S,/S, and S,/S, ratios of near unity were calculated (fig. 2). PMA concentration dependently increased the stimulation-induced release. With 10 FM PMA. this release was more than doubled. PME. on the other hand, was completely ineffective. Xylazine (2 I-(M) and FK 33-824 ( 1.36 PM) nearly halved the release of radioactivity and the effect of these drugs was completely antagonized by 10 PM PMA (fig. 2).

20mNl*-

66L-

Fig. PM

(A

six experiments

lo-

e-

a-

6-

6-

L-

L-

2-

2-

O-

o-

n-

and effect of 30 PM

dl

dz 0:3 OS ms

curves produced by btimulatkrn

of vasrl with l-s trains (15 Hz) of field pulses with increasing durarion. (A) Control The control curve cc?)was depressed hy _7 PM qlazine (A ) and this effect was antagonized by IO The conlrol curve (~7) was depressed by I.36 /.IM FK 33-824 (01 and this &fect was antagonized by IO FM (

PM \

( A) PMA. (0 ) PMA. The curves

W) and 30 PM

and 30 FM

IO-

2-

I. Slimulation-rrbponse

curve (a)

12 -

16I412-

tt -

lo-

( A ). (B)

shown in each panel were oht;lined

and the ;Issnci;lted S.E.

from the same vas;~ a( 1%min intervals. The symbols indicate means from

is shown by the veTlic;d hurs. Abscissas: field pulse duration

(ms). Ordinates:

force of contraction bnN).

11

7,

mN

A

mN

6-

6-

5-

5-

II-

I-

3-

3-

2-

2-

I-

I-

O-t

25

*

50

.

I

*

100 200 LOO

0

25

50

100 200 LOO @t

Fig. J. Concentration-response

curves for hethanecho!

scnce (

ments and the bar shws different

of

for 5 min: the rewlting

and S; /S,

[ions: CON are pi\en

rc!ease

It! /.LM

in the ab-

IO PM (

(13). The symbols hars indicate

concentration contraction

the corresponding

(FM!.

(mN!.

Ordinates:

S.E.s.

force

of

glycerol (DOG), in a concentration of 100 PM, also caused a slight (16%) but significant reduction in the contraction amplitude (fig. 3B). PME had no significant influence on noradrenaline-induced contractions (not shown). Application of bethanechol also conttacted the vas, but the maximum effect (6 IL0.5 mN, fig. 4) was smaller than the effect of noradrenaline. For bethanechol, an ECsr, of 118.6 PM (103.8-135.4 FM) was calculated. PMA, 1, 10 and 30 PM reduced the maximum effect of bethanechol by 15 (not shown), 23 and 47%, respectively (fig. 4A); the highest concentration of PMA also caused a small shift of the EC%, to 137.1 PM (122.6153.2 PM). DOG (100 PM) reduced the maximum effect of bethanechol by nearly 30% (fig. 4B) while PME was again ineffective (not shown). 3.4. CnCI, in depolarized

t’asa

Concentration-response curves for the contractile effect of CaCI, were obtained in vasa depolarized with a high concentration of KCI. Under control conditions, the maximum force induced by 4 mM CaCI, was 16.7 + 0.12 mN (fig. 5A) and the ECsr, was 0.98 mM (0.75I.26 mM). The alkaloid ryanodine, which depletes intracellular calcium stores, had no influence on the CaCl,-induced contractions (1 FM ryanodine, three preliminary experiments, data not shown). PMA (30 PM) depressed the maximum response to CaCI, by 59% (fig. 5A), but caused no significant shift of the ECs,,. PME, 30 ,xM, was completely ineffective (not shown). The influence of PMA on the contractile force was not altered in the presence of the calcium ionophore A 23187 (fig. 5B), but was greatly attenuated in vasa incubated for 1 h with 1 PM ouabain (fig. XI. In these vasa, the control curve for CaCI, was not changed, but PMA (30 PM) caused only a 20% reduction in the maximum developed force, which was siAnif-

59 20-

mN 7 A

mN 0 16

mN I&

16.

16-

6-

6-

C

0-e

. 0.25 0.5

mM

mH

. I

.

*

2

4

mM

Fig. 5. Influence of PMA (30 PM) on the concentration-response curve of calcium in depolarized vasa. (01 Controt,curve: t@) effect of PMA. (A) Effect of PMA alone. (B) Effect of PMA applied together with ionophore A 23187 (0.5 PM). (0 Effect of PMA in vasa pretreated with ouabain (1 PM) for I h. In (Cl the control curve was obtained in the presence of ouabain. The symbols show means from six experiments and the vertical bars indicate the associated S.E.s. Abscissas: concentration of CaCI, tmM). Ordinates: force of contraction tmN).

icantly less than the 59% observed in unpretreated vasa (U-test; P Q 0.05). Like PMA, the CaZC antagonist verapamil (0.03 PM) also caused a significant (55%) reduction in the CaCl,-induced contractions (fig. 6). However, the effect of verapamil was antagonized by the calcium ionophore A 23187 (0.5 PM).

4. Discussion In the present experiments PMA increased the release of noradrenaline from the field-stimulated mouse vas. The release of the cotransmitter ATP was not

20 mN

I 1

19

I 2 4 mM Fig. 6. The concentration-response curve for calcium in depolarized vasa (3) was depressed by verapamil 3X IO-” M (+a).Addition of 0.9 PM ionophore A 23187 antagonized the (effect ofverapamil t a ). The symbols show means from six experiments and the vertical bars indicate the associated S.E.s. Abscissas: concentration of CaCI, tmM). Ordinates: force of contraction (mN). 0.25 0.5

measured, but we assume that it was increased to a similar extent because ATP and noradrenaline are released from the same synaptic vesicles (Sneddon and Westfall, 1984). Transmitter release was not modified by PME. a drug which is structurally related to PMA but does not activate protein kinase C. Therefore, the most likely explanation for the effect of PMA is a stimulation of this enzyme. This confirms and extends earlier reports showing that PKC-activating phorbol esters increase the release of neurotransmitters from a variety of tissues (Wakade et al., 1985; Allgaier et al., 1987, 1988; Musgrave and Majewski, 1989). In our experiments PMA also antagonized the depressant influence of the cy,-adrenoceptor agonist xylazine and the p-receptor agonist FK 33-824 on the release of noradrenaline. This could indicate that the decrease in transmitter release caused by activation of prejunctional receptors is mediated by a down-regulation of PKC activity. Although there is evidence in favour of this interpretation (Brasch, 1991) Allgaier et al. (1987) suggest that prejunctional autoregulation and modulation of PKC activity are two independent mechanisms for the control of transmitter release. The present experiments were not designed to resolve this interesting question. As the neurotransmitters released by field stirnulation induced contractions of the vas via activation of postjunctional receptors, one would expect that PMA would modify the stimulation-response curves obtained in these experiments. The drug did indeed partly restore contractions when the stimulation--response curves were depressed by xylazine or FK 33-824, but no influence on the stimulation-response curve of unpretreated vasa was detected. Instead, PMA concentration dependently depressed the concentration-response

is indic:itcs thitt PMA has ;I postjunctional rclasing effect in the mouse \‘us. In the field stimulation cxperiments the op&ng prejunct~~~n~1~ (i.e. an increased re~casc of transmitters) and postjunctional (i.e. the decrease of transmitter-induced contractions) effects of PM.c\ seemed to cancel each other out, thus leaving the stimutation-response curve unchanged. The situation is different in vascular preparations, where the prejunctional and postjunctional effects of PMA are USUat& synergistic. However. the physiotogic~l significance of these findings is not clear, because it is unknown whether PKC can be stimulated selectively in neuronal or smooth muscle cells in vivo. The ~stjunctionat relaxing effect of PMA is probably caused by a stimulation of PKC. because (a) it was not shared by PME and (b) it was mimicked. though to a lesser extent. by the PKC stimulator DOG. The effective concentrations of PMA or DOG. however, were much higher than those usually needed for activation of PKC in other tissues. The reason for this discrepancy is not clear. It should be noted. however, that the EC,t, values for no~drenatine and bethanecholQ2 and.1 18 ~1M. respectively) were likewise much higher than those calculated for the effects of these drugs in other isolated organ preparations. A comparatively low sensitivity of the postjunctional receptors in the vas or a limited diffusion of the drugs from the organ bath to their postjunctional target sites are possihte explanations. Variable ~stjunctionat effects of PKC-activating phorbol esters have been described in previous publications. While contractions are usually induced in arteriaf preparations (Forder et al.. 1985; itoh and Lederis, 1988). uterine smooth muscle (Savinneau and Mironneau. 1990) and, according to one report, also in the rat vas deferens (Baraban et al.. 19851, relaxing effects have been observed in ileum preparations (Sasaguri and Watson. 1990) and tracheal smooth muscle (Menkes et al.. 1986: Hirst et al., 1989). Many explanations for the PKC-induced muscle relaxation have been suggested. A decreased receptor-effect coupling for adrenoceptors and muscarinic receptors may be responsible (Menkes et al., 1986; Lai et al., 1990). A decreased calcium influx via voltage-operated calcium channels fcapogrossi et al., 19901, a decreased intraceiluiar storage of calcium (Rogers et al., 19901, a decreased sensitivity of the contractile proteins for catcium (Katoh et al., 1983; Spedding, 1987) or an increased extrusion of calcium from the celi by different ion exchange mechanisms (Sasaguri and Watson, 1990) are other possibilities. The experiments in K+-depolarized vasa were done to differentiate between these possibilities. In these muscles, CaCl,-induced contractions are certainly not

mediated by a stimulation of adrenoceptors or muscarinic receptors. Instead, calcium ions enter the cells via voltage-controlled calcium channels, which are permanently open due to the tow membrane potential. A rclcasc of calcium from intracetlular stores apparently does not contribute to the CaCl,-induced contractions beca:!se the c~ltciunl-depleting agent ryanodine (Sutko ct a1., 1985; Meissner, 1986) caused no depression of the concentration-response curve. The depressant effect of the Ca’+ antagonist vcrapamil. caused by a reduced calcium influx, was an expected finding. PMA, but not PME, also decreased the contractile response to Ca’+. This indicates that activation of PKC does not interrupt receptor-effect coupling, but rather directly affects the cetiular handling of Ca’+. We considered it unnecessary, therefore. to investigate the influence of PMA on ATP-induced contractions mediated via purine receptors. While the effect of verapamil was antagonized by the Ca” ionophore A 23187, the effect of PMA was ionophore-resistant. The ionophore causes an influx of Ca” into the cell which is not dependent on Ca” channels and can thus compensate the block of the latter by verapamit. This shows that a reduced Ca” influx is not the cause for the ref$xant effect of PMA either. Earlier investigations (Greene and Lattimer, 1986; Yingst, 1988) have shown that activation of PKC can stimulate the Na+-Ki-ATPase. This could lead to a reduction in the intracellular Ca” concentration, due to activation of the Na+/Ca’+ exchange mechanism. The relaxant effect of phorboi esters in isolated ileum preparations has been exp&ed in this way (Sasaguri and Watson, 1990). The effect of PMA was attenuated by the Na+-K+-ATPase inhibitor ouabain in our experiments. We therefore propose that the activation of this enzyme plays a key role in the depression of contractility elici?;d by phorbofs Ic ‘the vas. However, these are preliminary results. MOII, dctailed experiments are needed to clarify, for instance, whether the Na+/Ca”’ exchanger, the Na’/H+ exchanger or othLr transport mechanisms are also involved before a definite mechanism of action can be proposed. In summary, the present experiments have shown that FMA, by activating PKC, increases the stimulation-induced release of transmitter in the mouse vas and simultaneousfy decreases the contractility of the smooth muscle.

Acknowledgements The authors wish to thank Mrs. G. v. Braun and Mrs. E. Obst for skiiiltii tcchnifal assistance. Mrs. M.-L. Stoke for drawing the figures and Mrs. G. Mundhenke for preparing the manuscript.

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