Essential Role of Myosin Light Chain Kinase in the Mechanism for Mgatp-Dependent Priming of Exocytosis in Adrenal Chromaffin Cells

The Journal of Neuroscience, December 1994, 74(12): 7695-7703

Essential Role of Myosin Light Chain Kinase in the Mechanism for MgATP-Dependent Priming of Exocytosis in Adrenal Chromaffin Cells

Konosuke Kumakura,’ Kimihito Sasaki,’ Takashi Sakurai,* Mica Ohara-lmaizumi,l Hiroaki Misonou,l Seiji Nakamura,* Yuzuru Matsuda,3 and Yoshiaki Nonomura* ‘Life Science Institute, Sophia University, Tokyo 102, *Department of Pharmacology, Faculty of Medicine, University of Tokyo, Tokyo 113, and 3The Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Tokyo 194, Japan

Ca*+-induced exocytosis in chromaffin cells now seems to goyne, 1990; Terbush and Holz, 1990; Wilson, 1990; Ohara- consist of at least two distinct steps: MgATP-dependent Ca2+- Imaizumi et al., 1992b), cytoplasmic proteins (Sarafian et al., enhanced priming of the secretory apparatus, and Ca2+-de- 1987; Ali et al., 1989; Morgan and Burgoyne, 1992; Nishizaki pendent MgATP-independent step that triggers exocytosis et al., 1992) and granule-associatedproteins (Darchen et al., (Bittner and Holz, 1992). Recently we found that a specific 1990; Pet-inet al., 199 1; Elferink et al., 1993). While the progress inhibitor of myosin light chain kinase (MLCK), wortmannin, by these studiesdoes not allow us yet to fully understand the inhibits Ca*+-induced catecholamine release from digitonin- biochemical events responsiblefor regulated exocytosis, mul- permeabilized chromaffin cells, suggesting an implication of tiple processesin Ca*+-dependentsecretion have been suggested MLCK in the mechanisms of Ca*+-induced exocytosis (Imai- recently. Holz and collaborators suggestedthat MgATP primed zumi et al., 1992b). To elucidate further the implication of secretion before Ca2+-triggering processin digitonin-permea- MLCK in the mechanism of exocytosis, we studied the ef- bilized cells, and that Caz+-evoked exocytosis from the primed fects of wortmannin and a peptide inhibitor (SM-1) corre- pool was independent of ATP (Holz et al., 1989; Bittner and sponding to the pseudosubstrate domain of MLCK on MgATP- Holz, 1992). Hay and Martin (1992) dissectedthe regulated dependent and MgATP-independent release in digitonin- secretion, in mechanically disrupted PC 12 cells, into MgATP- permeabilized chromaffin cells. Ca*+-induced exocytosis dependent priming and Ca2+-dependent MgATP-independent from the permeabilized cells in the presence of MgATP was triggering stages.In mast cells permeabilized by streptolysin-0, inhibited by both SM-1 and wortmannin. Inhibitory effect of ATP was required to maintain a high affinity of the response wortmannin on the rate of release induced by 10 PM Ca2+ in system for CaZ+and GTPyS, but was not required for the final the presence of MgATP was much prominent in the later stageof triggered exocytosis (Howell et al., 1989). phase (l-10 min), although the initial rate was also de- Recently we reported the inhibition by wortmannin of Ca2+- creased. SM-1 strongly inhibited ATP-dependent release induced catecholamine (CA) releasefrom digitonin-permeabil- without affecting Ca 2+-dependent ATP-independent release ized chromaffin cells as well as the releaseby ACh and high K+ at all. In addition, priming effect of MgATP that underlies from the intact cells (Ohara-Imaizumi et al., 1992b). Wort- Ca2+-dependent ATP-independent release was remarkably mannin inhibits myosin light chain kinase (MLCK) specifically reduced by both wortmannin and SM-1. These results sug- and potently (Nakanishi et al., 1992). Thus, we suggestedan gest that MLCK plays an essential role in ATP-dependent implication of MLCK in the mechanismsof Ca2+-induced exo- priming of Ca*+-induced exocytosis in chromaffin cells. cytosis. Possibleinvolvement of the contractile proteins or their [Key words: chromaffin cells, exocytosis, myosin light chain regulatory proteins in the mechanism of exocytosis in chro- kinase, ATP-dependent priming, wortmannin, MLCK pseu- maffin cells hasbeen proposedfrom earlier (Douglasand Rubin, dosubstrate domain peptide] 1964; Poisner and Trifaro, 1967; Trifaro, 1978). MLCK is present in adrenal medulla (Kanda et al., 198.5) and exogenous Recent studies on mechanismsof regulated secretion in chro- MLCK or secretagogueshave been shown to phosphorylate in- maffin cells have revealed possibleimplication of guanine nu- tracellular myosin light chain (MLC) in thesecells (Lee et al., cleotides and guanine nucleotide-binding proteins (Knight and 1987; Gutierrez et al., 1989). Despite thesefindings, physiolog- Baker, 1985; Bittner et al., 1986; Ohara-Imaizumi et al., 1992a; ical studies have failed to provide any evidence that suggests Vitale et al., 1993), various protein kinases(Morgan and Bur- the involvement of MLCK activation in the mechanismof exocytosis (Lee et al., 1987; Nakanishi et al., 1989) until our previous report (Ohara-Imaizumi, 1992b). Received Sept. 28, 1993; revised June 6, 1994; accepted June 9, 1994. To clarify the implication of MLCK activation in the multiple We thank Dr. T. Kanno and Ms. R. Nagata for help with Argus-IOOXa. This processesfor the regulated exocytosis, in this study we examined work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan to K.K. and to M.O.-I. The the effects of inhibition of MLCK activation on MgATP-de- Sasakawa Scientific Research Grant from The Japan Science Society to M.O.-I. pendent and MgATP-independent releaseof CA in digitonin- is also acknowledged. permeabilized chromaffin cells. We particularly used a potent Correspondence should be addressed to Konosuke Kumakura, Ph.D., Life Sci- ence Institute, Sophia University, 7-1, Kioi-cho, Chiyoda-ku, Tokyo 102, Japan. inhibiting peptide, SM- 1, that correspondsto the pseudosub- Copyright 0 1994 Society for Neuroscience 0270-6474/94/147695-09$05.00/O strate domain of MLCK required for autoinhibition (Kargacin 7696 Kumakura et al. * MLCK and MgATP-Dependent Priming of Exocytosis et al., 1990) and other inhibiting peptides in addition to wort- rescence ratio (340 nm:380 nm) using the equation described by mannin. We also studied the localization of myosin II for un- Grynkiewicz et al. (1985). derstanding the site of MLCK implication. Preparation of antibody and immunoblotting. Myosin heavy chain was purified from chicken gizzard smooth muscle according to the method previously described (Nakamura and Nonomura, 1984). Preparation and characteristics of polyclonal anti-myosin II IgG will be described Materials and Methods in details elsewhere (Ogiwara, unpublished observations). This antibody Primary cultures. Adrenal chromaffin cells were isolated from fresh specifically and strongly reacted with mammalian nonmuscle myosin bovine adrenal glands by collagenase digestion, and cells were purified II heavy chain as well as smooth muscle one (see Results and Fig. lOA). by differential plating as previously described (Kumakura et al., 1986). For immunoblotting, cultured chromaffin cells were collected after The purified chromaffin cells were plated (3 x 10s cells/ml) on collagen- rinsed with PBS, and homogenized by a small Grass-homogenizer in coated 16 mm diameter dishes (Nunc), and were maintained in mono- the solution containing 50 mM KCl, 25 mM Tris-HCl (pH 7.6), 50 PM layer culture in Dulbecco’s modified Eagle’s Medium (DMEM; GIBCO) DFP, 50 PM NaN,, and 1 mM MgCl,. The homogenate was centrifuged supplemented with 10% fetal calf serum (Boehringer Mannheim) as was in an Eppendorf tube at 3500 x g for 10 min. The supematant was described previously (Ohara-Imaizumi et al., 1992a). All the experi- centrifuged at 16,000 x g for 90 min, and the precipitate solved in SDS ments were performed with the cells in culture for 3-10 d. solution was used as microsome-rich fraction. Proteins resolved by SDS- Permeabilization of cells. The chromaffin cells in monolayer were PAGE were transferred to nitrocellulose sheets (Schleicher and Schuell, permeabilized by in&bating for 4 min at 37°C in a buffer (P-buffer) Inc.; 0.25 pm), and after staining with Ponceau S and photographed, the samples were reacted with anti myosin II IgG (diluted 1:500) and containing 140 mM Na-dutamate. 20 mM PIPES, 5 mM glucose. 5 mM EGTA, aid 0.02 mM di&onin (&lbiochem) adjhsted pfi to 6.80 with HRP-conjugated anti-rabbit IgG according to the conventional method, NaOH (Ohara-Imaizumi, 1992a). This permeabilizing buffer was sub- and stained with 4-chloro-naphthyl phosphate. sequently replaced with a buffer for a secretion experiment. Immunofluorescent microscopy. Chromaffin cells cultured on cover- Secretion experiments. The release of CA from the digitonin-per- slips were fixed with 0.8% paraformaldehyde in PBS for 1 min and, meabilized cells was measured as previously described (Ohara-Imai- after washing with PBS, with 100% acetone for 5 min at room tem- zumi, et al., 1992a). At the end of permeabilization, P-buffer was re- perature. The cells were treated with 1% Triton X- 100 in PBS for 5 min placed with a buffer (S-buffer) containing (in mM) 140 Na-glutamate, at room temperature, rinsed in PBS, and then incubated with the block- 20 piperazine-N,N’-bis(2-etbanesulfonic acid) (PIPES), 5 glucose, 5 ing solution containing 2.5% bovine serum albumin and 2% chicken MgSO,, 5 ATP, and 5 EGTA (without or with various amount of CaCl,), serum for 30 min. Subsequently, the cells were incubated with anti with pH adjusted to 6.80 with NaOH. The cells were incubated for an myosin II IgG (diluted 1:400) for 3 hr at 37°C. After rinsing in 400 mM additional 6 min and the medium was collected for measurement of M&l, and 20 mM Tris-HCl (pH 7.6) and PBS, the cells were incubated Ca*+ -evoked CA release. Calcium concentrations in Ca2+ -EGTA buffers fo; 1 hr at 37°C with FITC-conjugated anti rabbit IgG in the blocking were calculated according to Nanninaa and Kemuen (197 1). For the solution. The samples were mounted in 5% DABCO in PBS/glycerol measurement of MgATP&dependentrelease, the cells ‘were incubated (1: 1) and visualized under Nikon FEX-A fluorescent microscopy. for l-2 min in S-buffer containing 100 PM Ca2+ without MgSO, and ATP. For MgATP-dependent release, the concentration of free Ca2+ in S-buffer was adjusted to 10 PM, and the cells were incubated for 6 min Results according to Bittner and Holz (1992) in the presence of 5 mM MgSO, Inhibition of CA release by wortmannin and 5 mM ATP. The release of endogenous CA from intact chromaffin Preincubation of adrenal chromaffin cells with wortmannin pro- cells in monolayer culture was measured as previously described (Ohara- Imaizumi et al., 1992a). Briefly, the cells were stimulated in Locke’s duced a marked decreasein CA releaseevoked by ACh from solution for 3 min following the preincubation for 30 min at 30°C. the intact cells (Fig. 1). The inhibitory effect of wortmannin on Released CA and cellular CA were extracted with 0.4 M perchloric acid ACh-evoked releasewas dose dependent, and a significant in- and assayed by HPLC (Kumakura et al., 1986). hibition was observed at concentrations above 0.3 PM. The ap- Priming effectofA TP. To examine the priming effect of ATP, priming incubation was done simultaneously with permeabilization for 4 min parent IC,, value for the inhibition was approximately 2 PM in the permeabilizing buffer (P-buffer) containing 5 mM MgSO,, with that was consistent with IC,b for the inhibition of high-K+- or without 5 mM ATP. The buffer was subsequently replaced with S-buf- evoked and Ca*+-inducedrelease (Ohara-Imaizumi et al., 1992b). fer without ATP, and MgATP-independent release was measured. Wortmannin at 10 PM suppressedACh-evoked releasealmost Wortmannin and MLCK-inhibiting peptides. Wortmannin was pre- completely as was observed for high-K+-evoked and Ca*+-in- pared as previously described (Nakanishi et al., 1992). SM-1 was syn- thesized and purified according to Wysolmerski and Lagunoff (199 1). duced release in our previous report (Ohara-Imaizumi et al., SM-1 has the following. sequence: Ala-Lys-Lys-Leu-Ser-Lys-Asp-Arg- 1992b). Wortmannin did not affect either the basal releaseor Met-Lys-Lys-Tyr-Met-&a-Arg-Arg- Lys-Trp-Gin-Lys-Thr-Gly (MLCC the noradrenaline:adrenalineratio in the releasedCA (data not 480-501). Myosin kinase-inhibiting peptide (MKIP; Lys-Lys-Arg-Ala- shown). The releasein the absenceof CaZ+ in the digitonin- Ala-Arg-Ala-Thr-Ser-NH,; analog of MLC,,, 1 l-l 9) and N-terminal acetylated MKIP (MKIPAc) were prepared according to Hunt et al. permeabilized cellswas not affected by wortmannin (4.4 + 0.2% (1989). A pseudosubstrate peptide for &ILCK (K-MLC l l-23), and for and 4.1 + 0.2% in 10 PM wortmannin-untreated and -treated orotein kinase C (PKC). ISer251PKC( 19-3 1). were nurchased from Pen- cells, respectively). Wortmannin did not affect the cellular con- jnsula Laboratories (B&nont, ?A). . ” - tent of CA at all (data not shown). These resultstogether with To examine the effects of wortmannin, 30 min preincubation was our previous findings suggestthat the inhibitory action of wort- done in the presence of the drug prior to the digitonin permeabilization. The DeDtide inhibitors were added in the nemeabilizina P-buffer and mannin on the evoked CA releasefrom intact cellsand on Caz+- also in S-buffer for some experiments (see figure captions). induced releasefrom the permeabilized cells is mediated by the Monitoring of intracellular calcium. For the monitoring of intracel- samemechanism. lular calcium, the cells cultured on a coverslips coated with Cell-Tak (Biopolymers, Farmington, CT) were loaded with 2 PM fura-2/AM in Time dependency of the inhibitory action of wortmannin DMEM for 1 hr at 37°C using a Sykus-Moore culture chamber, and rinsed three times with Locke’s solution. Fura-2-loaded cells were in- Since the inhibition of purified MLCK by wortmannin in vitro cubated for another 30 min in the presence or absence of wortmannin is dependent on the preincubation time that is required for the prior to Ca*+ monitoring. The cells were superfused (2 ml/min) with interaction of the drug with the enzyme (Nakanishi et al., 1992), Locke’s solution at 30°C for fluorescence measurement. Digital imaging we examined the effect of preincubation time on susceptibility of fura- fluorescence was carried out with an inverted microscope (TMD-2, Nikon, Tokyo, Japan) and a digital image processor (Argus- of Ca*+-induced releasefrom the permeabilized cells. When the lOO/Ca, Hamamatsu Photonics, Hamamatsu, Japan). The concentra- cells were preincubated in the presenceof 10 PM wortmannin tion of intracellular free Ca*+ ([Ca*+],) was estimated from the fluo- for a different time prior to the permeabilization, clear inhibition The Journal of Neuroscience, December 1994, 14(12) 7697

j -0--a*+ OHM

0 .l 1 10 01 I I I 0 10 20 30 [Wottmannin] /pM

Figure 1. Dose-response curve for wortmannin inhibition of ACh- Incubation Time / min evoked from intact chromaffin cells. CA release from the intact cells was measured by stimulating the cells with 1O-4 M ACh for 3 min Figure 2. Requirementof preincubationtime for wortmannininhi- following the ureincubation for 30 min at 30°C in the nresence or absence bition of Ca2+-evokedCA releasefrom the permeabilizedcells. The of increasing-concentrations of wortmannin. The amounts of released cellswere preincubated with 10PM wortmanninfor the indicatedtimes CA are expressed as the percentage of total cellular content. Sponta- at 30°Cprior to permeabilization.Permeabilization was done with 10 neously released CA was measured for each group, and the mean value PM digitoninfor 10min in this particularexperiment. The permeabilized for each group was subtracted from each evoked release. Data are the cellswere stimulated with 10PM Ca*+ for 10min to measureCA release. mean ? SEM of six determinations from individual wells. The amountsof releasedCA are expressedas the percentageof total cellular content. Data are the mean + SEM of eightdeterminations from individual wells.Note that wortmannindid not affect the basal releasein the absenceof Ca2+. of Ca2+-induced release was observed only after the preincubation for more than 15 min, and preincubation for 30 min was required for nearly maximal inhibition (Fig. 2). This time de- were also examined. As was shown in Figure 4, SM- 1 inhibited pendency was similar to that observed for the interaction of Ca2+-inducedrelease from the permeabilized cells by approx- wortmannin with MLCK in vitro (Nakanishi et al., 1992). imately 25% at 100 PM, 50% at 370 PM, and 90% at 500 PM. K-MLC 1 l-23 causedapproximately 20% inhibition at 500 PM. Wortmannin does not inhibit Ca2+ influx MKIP was much weaker than SM- 1 in inhibiting Ca2+-induced In human platelet, our group found an effective blockade by release.The inhibition by 500 PM MKIP was about 15%, al- wortmannin of a new type of Ca2+ channel that acted in the though it was statistically insignificant, and such an inhibition delayed phase of the response to thrombin stimulation (Hash- was not observed with the inactive analog MKIPAc. In contrast, imoto et al., 1992) Thus, it was necessary to check the effect of [SedS]PKC(19-31) at 100 PM did not affect the Ca2+-induced wortmannin on CaZ+ entry in the present study. releasefrom the permeabilized cells at all in good agreement Stimulation of fura-2-loaded intact cells with carbamylcho- with a previous report (Terbush and Holz, 1990). None of these line (CCh; 2 x 1O-4 M) by adding 100 ~1 of CCh into superfusing peptides significantly affected CA releasein the absenceof CaZ+. chamber produced a transient increase of [Ca*+], to 482 f 43 It has been shown that MKIP is much lesspotent than SM-1 nM (n = 6). The same concentration of CCh produced [Ca*+] in inhibiting Ca*+- and ATP-induced retraction of permeabil- increase to 539 -t 71 nM (n = 6) in wortmannin-treated cells. ized endothelial cells (Wysolmerski and Lagunoff, 1991). The These concentrations of [Ca2+], are comparable to those required weak inhibitory action of MKIP seemsto be due to the fact that for the diphosphorylation of MLC by MLCK in vitro (Itoh et this peptide is lesspotent in inhibiting MLCK in the permea- al., 1992). Representative fura- signals are shown in Figure 3. bilized chromaffin cells as well. These results suggest that wortmannin acted on the downstream These results, especiallyeffective inhibition by SM-1 of Ca2+- [Ca2+], increase or on the prerequisite process for Ca2+-triggering induced releasefrom the permeabilized cells, strongly suggest events in CA release in intact cells. that the inhibitory action of wortmannin resulted from the inhibition of MLCK activation. Efects of SM-1 and other peptide inhibitors To clarify that the inhibitory action of wortmannin is attrib- Efects of wortmannin on time courseof Ca2+-inducedrelease utable to its inhibition of MLCK, we examined the effects of In the time courseof Ca*+-induced releasefrom permeabilized various synthetic peptides that are known to inhibit MLCK: chromaffin cells, it hasbeen shown that the initial rate of release SM- 1, K-MLC 1 l-23, MKIP, and its inactive derivatives MKI- during first l-2 min is not altered by the absenceand presence PAC. The effects of pseudosubstrate inhibitory peptides for PKC of MgATP while MgATP is required for the releaseto continue 7696 Kumakura et al. l MLCK and MgATP-Dependent Priming of Exocytosis

614

z 463 E . r 312 B 161

120 240 360 480 Time/s e SM-1 772 d MKIP ,Wortmannin-treated d-- MKIP-AC - K-MLC 581 II 5 I I . k&= 391 n [ peptides ] /PM E 2oc Figure 4. Effectsof SM-1, K-MLC 1l-23, MKIP, and MKIP-AC on Ca2+-evokedCA releasefrom the permeabilizedcells. The cellswere 1 permeabilizedwith 20 PMdigitonin for 4 min,and then stimulatedwith 10PM Ca2+for additional6 min at 30°C.Peptide inhibitors were present during wholeprocedure at the indicatedconcentration. The amounts I 120 240 360 480 of releasedCA areexpressed as the percentageof total cellularcontent. Time Is The amountsof releasedCA are expressedas the percentageof total 4 cellularcontent. Amount of CA releasedin the absenceof Ca2+was Figure measuredfor eachgroup, and the meanvalue for eachgroup was sub- 3. Effect of wortmanninon CCh-evokedincrease of intracel- tractedfrom each evoked release. Data are the mean ? SEM of at least lular Ca*+@ZzZ+jJ. Ca2+ signals of a singlecell weredetected with a six determinationsfrom individual wells.None of the peptidesaffected video-imagingsystem using fura- in control cells(top) andwortman- CA releasein the absenceof Ca*+. nin-treatedcells (bottom). The cellswere preincubated with or without 10 I.IMwortmannin for 30 min after loadingof fura-2. Repetitivestim- ulation with 2 x 10m4M CChwas given in the superfusingchamber at the pointsindicated by arrows. Responsesin A and B are the represen- 1992). These results clearly suggesta requirement of MLCK tative fura- signals.Mean valuesof the increased[Cal+], in control activation for the continuous releasethat is sustainedby MgATP. and wortmannin-treatedcells were 482 + 43 nM (n = 6) and 539 ? 71 nM (n = 6), respectively. Efects of wortrnannin and SM-1 on ATP-dependent and ATP-independent release thereafter (Holz et al., 1989). Therefore, we studied the effects To obtain further evidence for requirement of MLCK for ATP- of three different concentrations (1,3, and 10 KM)of wortmannin dependent release,we examined the effectsof wortmannin and on the time course of CA releaseevoked by 10 PM Ca2+ from SM-1 on ATP-independent and ATP-dependent releasefrom the permeabilized cells. As shown in Figure 5, the time course the permeabilized cells. When the cells were pretreated with 10 of Ca2+-induced releasein control cells apparently consistedof PM wortmannin, both ATP-independent releaseand ATP-de- kinetically distinct three components: (1) the initial phasefor pendent releasewere decreasedapproximately by 78% and by the first 1 min, (2) the second phasefrom 1 to 5 min, (3) the 90%, respectively (Fig. 6). In contrast to wortmannin, treatment third phaseafter 5 min. Wortmannin reduced the releasein the of the cells with 370 PM SM- 1 for 4 min prior to Ca*+ challenge initial phaseto about one-third of control, without any dose caused 55% inhibition of ATP-dependent releasewithout any dependency in both the extent and secretion rate. In contrast, inhibition of ATP-independent release.Another inhibiting pep- effectsof wortmannin on the later phasewere much prominent. tide, MKIP, also decreasedATP-dependent releasebut slightly In the cells treated with 1 PM wortmannin, the releasein the (Fig. 7). The inhibition by SM- 1 and MKIP at this concentration second and third phasepersisted up to 10 min though the se- were close to that observed in Figure 4. cretion rate was reduced. Wormannin at 3 PM causedcessation The effectsof wortmannin and of SM- 1 on ATP-independent of the releaseat 2 min without further decreasein the rate of releasewere different. One possibility seemsto be that a dec- this shortenedsecond phase. Wortmannin at 10 MM almostcom- rement in the population of the primed granulesmight be caused pletely abolished both the second and third phase; thus, the during the longer preincubation time (30 min) with wortmannin release nearly ceasedat the end of the initial phase. Conse- but not within 4 min of preincubation with SM-1, since ATP- quently, the duration and final extent of the releasewere de- dependent priming hasbeen shownto be readily reversible (Holz creasedby wortmannin with clear dosedependency that is com- et al., 1989; Hay and Martin, 1992). However, due to the ex- parable to that we reported previously (Ohara-Imaizumi et al., perimental limitation with digitonin-permeabilized cells, we The Journal of Neuroscience, December 1994, 74(12) 7699

__o-- Control __c_ Wort.lpM 0 Control - Wort.3pM El Wottmannin treated _+__ Wort.1 OpM

1.5min (-ATP) 6min (+ATP) Oi Ca2+ 100pM 0 2 4 6 8 10 Ca2+ 10pM Figure 6. Effects of wortmannin on ATP-independent and ATP-de- Time / min pendent CA release from the permeabilized cells. The cells were preincubated at 30°C with or without 10 PM wortmannin for 30 min prior Figure 5. Effects of wortmannin on the time course of @+-evoked to permeabilization in the absence of MgATP. Then, ATP-independent CA release from the permeabilized cells. The cells were preincubated (--AU’) and ATP-dependent (+ATP) release was measured by stimu- with or without different concentration of wortmannin for 30 min prior lating with Ca2+ at the indicated concentration. The amounts of released to permeabilization. The permeabilized cells were stimulated with 10 CA are expressed as the percentage of total cellular content. Each value PM CaZ+ in the presence of MgATP for the indicated times. The amounts was calculated as was described in Figure 4. Data are the mean f SEM of released CA are expressed as the percentage of total cellular content. of eight determinations from individual wells. Each value was calculated as was described in Figure 4. Data are the mean + SEM of at least six determinations from individual wells. could not examine the effect of longer treatment with SM- 1. The secretory activity is known to run down rapidly after dig- 12 q Ca2+ 100pM x 1.5min (-ATP) itonin permeabilization. The response to 100 PM Ca2+ at 5 min 0 Ca2+ 10pM x 6min (+ATP) after 4 min of digitonin permeabilization decreased to 17% of 10 the responsewithout an interval, and the responsewas almost E I lost after 15 min interval (data not shown). The inhibition of rT ATP-independent release by tiortmannin should underlie its 70% inhibition of the initial releasein the time course(Fig. 5) 1 that was not dose dependent. This may imply alternatively that the inhibition of ATP-independent releaseby wortmannin is due to a mechanism other than MLCK inhibition. Whatever the mechanismof the inhibition of ATP-independent release by wortmannin that remained uncertain, theseresults together with the results in time course (Fig. 5) indicate a requirement of MLCK for the continuous releasesustained by MgATP. Wortmannin, S&f-l, and K-MLC 11-23 inhibit priming :.:.::::y.:::::::::.:...... :.: :.:.:: ::.: : : action of ATP ):.:::::.:_L:;:.. ::.y Since ATP-dependent releasefrom the permeabilized cells has -Ill been suggestedto be attributable to the action of ATP to prime Control MKIP SM-1 the secretory machinery (Bittner and Holz, 1992), we examined Figure 7. Effects of SM- 1 and MKIP on ATP-independent and ATP- the effect of wortmannin and MLCK-inhibiting peptides on dependent CA release from the permeabilized cells. SM- 1 or MKIP was priming action of ATP. As shown in Figure 8, priming with added at 370 PM during 4 min of permeabilization in the absence of ATP for 4 min remarkably increasedboth the initial rate and MgATP. ATP-independent (-A TP) and ATP-dependent (+A TP) re- final extent of ATP-independent release.Pretreatment of the lease were measured by stimulating with Ca*+ at the indicated concen- cells with wortmannin caused a great decreasein the priming tration. The amount of released CA is expressed as the percentage of total cellular content. Each value was calculated as was described in effects of ATP, in addition to a decreasein ATP-independent Figure 4. Data are the mean + SEM of eight determinations from releasein the unprimed cells (Fig. 8). SM-1 at 100 PM caused individual wells. 7700 Kumakura et al. l MLCK and MgATP-Dependent Priming of Exocytosis

--O- Primed B d Unprimed 6- d- Control __cl- Wott.Primed d Wart.-treated 1 + WottAJnprimed

1 1 Time / min Time / min

Figure 8. Effect of wortmannin (Work) on ATP-dependent priming of CA release in the permeabilized cells. The cells were preincubated with or without 10 PM wortmannin for 30 min prior to permeabilization. Priming incubation with 5 mM MgATP was done simultaneously with 4 min of permeabilization. Stimulation with 100 MM Ca*+ was done for the indicated times in the absence of ATP. Amount of CA released in the absence of Ca2+ was subtracted as described in Figure 4. The amount of released CA is expressed as the percentage of total cellular content. Data are the mean k SEM of eight determinations from individual wells.

36% inhibition of the priming effect of ATP, and K-MLC 1 l- 23 at 500 PM caused30% inhibition (Fig. 9). The inhibition by Discussion SM-1 at this lower concentration was more pronounced than Is MLCK involved in the mechanismof exocytosis? that observed in Figure 4. These results suggestthat activation Our results suggestthat MLCK is involved in the mechanisms of MLCK is an essentialstep for the priming of secretion by of Ca*+-induced exocytosis in chromaffin cellsas follows. Wort- MgATP. mannin inhibited ACh-evoked releasefrom the intact cells with IC,, of approximately 2 I.LM (Fig. l), which is consistent with Immunoblotting and immunojluorescentlocalization of IC,, for Ca2+-induced release in permeabilized cells (Ohara- myosin Imaizumi et al., 1992b). The preincubation time required for The myosin light chain kinasesare known to possessan absolute clear inhibition by wortmannin (Fig. 2) was very similar to that specificity for myosin light chains (for review, seeKennelly and required for the interaction of this drug with MLCK in vitro Krebs, 1991). Therefore, we studied the cellular distribution of (Nakanishi et al., 1992). Wortmannin inhibits MLCK activity antigen site for myosin antibodies in order to understand the acting irreversibly at or near the catalytic site of the enzyme, site of MLCK implication. The anti-myosin II IgG usedin this whereas it had no effect on CAMP-dependent protein kinase, experiments specifically recognized a single band in immuno- cGMP-dependent protein kinase, Caz+/calmodulin-dependent blotting of the microsome-rich fraction. The position slightly protein kinase II, and little effect on PKC (Nakanishi et al., lower than that of chicken gizzard myosin heavy chain (200 1992). Although we found very recently that wortmannin also kDa) was very reasonableas nonmuscle myosin II heavy chain inhibits phosphatidylinositol 3kinase (Yano et al., 1993), its (Fig. 1OA). Strong immunofluorescencewas observed mainly at IC,, (2-3 nM) is extremely lower than that for the inhibition of the subplasmalemmalregion (Fig. 10B) as both intense rings MLCK (Nakanishi et al., 1992) and of CA releaseobserved and patched distribution. The weak immunofluorescenceob- here. Hence, the inhibition of CA releaseby wortmannin ob- servedin the cytoplasmic area could be interpreted as either the served here seemsto be attributable to its inhibitory action on subplasmalemmaldistribution of myosin beneath the area or MLCK. the cytoplasmic distribution as has been reported (Gutierrez et Among MLCK-inhibiting peptides, SM- 1 at 500 PM produced al., 1989). These immunoblotting and immunofluorescentdis- almost complete inhibition of Ca2+-induced releasefrom the tributions of myosin may suggestthat myosin II is the most permeabilizedcells. Another MLCK-inhibiting peptide, K-MLC probable substrate,situated at the site of secretion, for MLCK 1l-23, caused a significant inhibition, while MKIP was much involved in ATP-dependent priming mechanisms.Hence, phos- weaker (Fig. 4). SM-1 is known to be extremely potent (Foster phorylation of myosin II in the subplasmalemmalregion by et al., 1990) and highly specific inhibitor for MLCK (Kargacin MLCK activation may be a part or prerequisite of the priming et al., 1990). The primary structure of nonmuscle MLCK and process. that of smooth muscle MLCK are identical except for N-ter- The Journal of Neuroscience, December 1994, 14(12) 7701

6 high as 100 PM was required for clear inhibition even with an 1 intracellular injection (Akasu et al., 1993). Excef ’ presenceof endogenoussubstrate and possibleleakage of protein phosphatase from cytosol in our experimental condition might explain the requirement of this high concentration. MKIP was much less potent than SM-1 as was reported for Ca2+- and ATP- induced retraction of permeabilized endothelial cells (Wysol- merski and Lagunoff, 1991). Anti-calmodulin antibody has been shown to inhibit high- K+-evoked and ACh-evoked CA releasewhen introduced into chromaffin cells (Kenigsbergand Trifaro, 1985). This seemsto be consistent with the present findings, since MLCK is a Ca*+/ calmodulin-activated enzyme. How is MLCK involved? Ca2+-induced releaseof CA from the permeabilized chromaffin cells or from the semiintact PC 12 cells has been resolved into at least two distinct stages:MgATP-dependent priming of secretion, and Ca*+-triggered ATP-independent releasefrom the primed pool (Bittner and Holz, 1992; Hay and Martin, 1992). In Ca*+-induced release from digitonin-permeabilized chromaffin cells, the initial release for l-2 min reflects ATP-independent releasefrom the primed pool, and the releasethereafter control Wort. SM-1 K-MLC is sustainedby ATP-dependent priming (Holz et al., 1989; Bitt- Figure 9. Effects of SM- 1 and K-MLC 1 l-23 on ATP-dependent prim- ner and Holz, 1992). The former of thesetwo componentscan ing of CA release in the permeabilized cells. Priming incubation with be distinguished as the releaseevoked by 100 PM Caz+ in the 5 mM MgATP was done simultaneously with 4 min of permeabilization absenceof ATP that terminates by l-2 min, thus termed ATP- in the presence or absence of 100 PM SM- 1 or of 500 PM K-MLC ll- independentrelease. The latter one is distinguishedas the release 23 (K-MLC). MgATP-independent release for 1.5 min was then measured in the absence of the inhibiting peptide. Value is the extent of evoked by 10 MM Caz+ in the presenceof MgATP that continues increase by priming. Mean value of the release in unprimed cells was for 10-12 min, thus termed ATP-dependent release. subtracted from individual value of the release in primed cells. The In addition, the priming effect of ATP can be observed as peptides affected neither the basal release nor the release from the un- such that preincubation of permeabilized cells with MgATP primed cells. Data are the mean f SEM of eight determinations from enhancesCaZ+ -dependent releaseduring a subsequentincuba- individual wells. Inhibition by wortmannin (Wort.) detected at 2 min in Figure 8 was also shown. tion in the absenceof MgATP either in chromaffin cells or in PC12 cells (Holz et al., 1989; Hay and Martin, 1992). Similar multiple processesof Caz+-dependent release in chromaffin cells minal region (Shoemakeret al., 1990). Therefore, the inhibition were suggestedvery recently from kinetic analysisof the mem- of CA releaseby SM- 1 in the present results can be interpreted brane capacitance and intracellular Ca2+ concentration mea- as a result from specific inhibition of MLCK. Significant inhi- sured simultaneously (Neher and Zucker, 1993). bition by K-MLC 11-23 supports this interpretation. The IC,, The time course of Ca2+-induced releasefrom the permea- of SM- 1 for this inhibition seemsconsiderably high. However, bilized cells in this study consistedof kinetically distinct three SM-1 was recently shown to inhibit voltage-dependent potas- components.Wortmannin differently affected thesethree stages, sium currents in sympathetic neurons, and a concentration as and the effects of wortmannin were more pronounced on the

B A a

Figure10. A, Immunoblotting results by anti-myosin II IgG: a, protein staining by Ponceau staining; b, immuno- staining; 1, chicken gizzard myosin; 2, membrane rich fraction from chromaffin cells.Arrowhead indicates the position of 200 kDa molecular weiaht of myosin II heavy chain. Double&row- headindicates the position of 100 kDa. The band at 100 kDa in protein staining shows perhaps the Ca-ATPase in membrane fraction. B, Distribution of myosin in chromaffin cells detected by Im- munofluorescent method. 7702 Kumakura et al. l MLCK and MgATP-Dependent Priming of Exocytosis release during the second and third phases than on the initial by intracellular injection of SM- 1 and anti-myosin antibodies release. Wortmannin reduced dose dependently both the rate was clearly demonstrated by detecting EPSP in response to pre- and duration of the release in the second and third phases (Fig. synaptic stimulation. In that study, the initial burst of the trans- 5). Consistently, ATP-dependent release was inhibited by SM- mitter release seems to be resistant to wortmannin while the 1, while ATP-independent release was not affected at all (Fig. sustained release is effectively blocked by the inhibitor. Thus, 7). The priming by MgATP that underlies ATP-dependent re- MLCK seems to be essential for priming of exocytosis also in lease was inhibited remarkably by wortmannin, and significantly sympathetic nerve terminals (S. Mochida, H. Kobayashi, Y. by either SM- 1 or K-MLC 1 l-23 (Figs. 8,9). All of these results Matsuda, Y. Yuda, K. Muramoto, and Y. Nonomura, unpub- clearly suggest that MLCK has an essential role in ATP-depen- lished observation). dent priming pathway. In conclusion, our results strongly suggest an essential role of A question arises, then, whether phosphorylation of MLC,, MLCK for priming of exocytosis. Most probably phosphory- mediates the putative role of MLCK or not in the regulated lation of MLC,, by MLCK activation plays an essential role in exocytosis. The catalytic and calmodulin regulatory segments ATP-dependent priming of exocytotic machinery. It has to be of nonmuscle MLCK are virtually identical to that of smooth determined whether phosphorylation of MLC,, is essential as muscle MLCK (Shoemaker et al., 1990). The sequences around the intrinsic mechanism of priming or as the microenvironment the phosphorylation sites of smooth muscle and nonmuscle my- to maintain priming. Reevaluation of the role of MLCK in Ca2+ - osin II of higher eukaryotes are highly conserved (for review, dependent exocytosis is desirable in terms of ATP-dependent see Tan et al., 1992). These findings imply that also nonmuscle priming mechanism. MLCK may possess an absolute specificity for MLC,,. Phos- phorylation of MLC,, by activated MLCK regulates the actin- myosin interaction in contraction of smooth muscle (for review, References see Adelstein and Eisenberg, 1980). The phosphorylated adrenal Adelstein RS, Eisenberg E (1980) Regulation and kinetics of actin- medullary myosin has been shown to enhance the actin-acti- myosin-ATP interaction. Annu Rev Biochem 49:921-956. Akasu K, Ito M, Nakano T, Schneider CR, Simmons MA, Tanaka T, vated Mg*+-ATPase activity (Kanda et al., 1985). However, an Tokimasa T, Yoshida M (1993) Myosin light chain kinase occurs implication of actin-myosin interaction in the putative role of in bullfrog sympathetic neurons and may modulate voltage-depen- MLCK for ATP-dependent priming remains uncertain for two dent potassium currents. Neuron 11: l-l 3. reasons. First, the role of actin as a barrier to the movement of Ali SM, Geisow MJ, Burgoyne RD (1989) A role for calpactin in calcium-dependent exocytosis in adrenal chromalhn cells. Nature 340: secretory vesicles to exocytotic sites has been suggested (Aunis 313-315. and Bader, 1988; Vitale et al., 199 1; reviewed by Trifaro et al., Aunis D, Bader MF (1988) The cytoskeleton as a barrier to exocytosis 1992). If actin-myosin interaction is involved in ATP-depen- in secretory cells. J Exp Biol 139:253-266. dent priming, therefore, the actin should be distinct from fila- Bittner MA, Holz RW (1992) Kinetic analysis of secretion from per- mentous actin that acts as a barrier. One may speculate the meabilized adrenal chromaffin cells reveals distinct components. J Biol Chem 267:16219-16225. interaction of myosin with oligomeric actin molecules. In this Bittner MA. Holz RW. Neubiz RR (1986) Guanine nucleotide effects regard, electron microscopic studies by quick freezing and deep on catecholamine secretion from%digitonin-permeabilized adrenal etching have demonstrated that exocytosis occurred more fre- chromaffin cells. 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Biochem J 264:589-596. It is likely that priming process is regulated through multiple Hashimoto Y, Ogihara A, Nakanishi S, Matsuda Y, Kurokawa K, Non- reactions. From this point of view, MLCK or phosphorylation omura Y (1992) Two thrombin-activated Ca2+ channels in human of MLC,, may be one of the essential components for priming. platelets. J Biol Chem 267:17078-1708 1. The putative role of MLCK in the mechanism of exocytosis Hay JC, Martin TF (1992) Resolution of regulated secretion into se- quential MgATP-dependent and calcium-dependent stages mediated suggested here might be quite common and fundamental in by distinct cytosolic proteins. J Cell Biol 119:139-l 5 1. different neurosecretory cells, since we now know that wort- Hay JC, Martin TF (1993) Phosphatidylinositol transfer protein re- mannin inhibits secretory function in the various types of cells quired for ATP-dependent priming of Ca*+-activated secretion. Na- such as rat basophilic leukemia cells, human basophils (Kitani ture 366~572-575. et al., 1992), PC 12 cells (Inoue and Kumakura, 1992), and cul- Holz RW, Bittner MA, Peppers SC, Senter RA, Eberhard DA (1989) MgATP-independent and MgATP-dependent exocytosis. Evidence tured rat sympathetic ganglion (Mochida et al., 1992; Muramoto that MgATP primes adrenal chromaffin cells to undergo exocytosis. et al., 1993). In cultured rat sympathetic ganglion, particularly, J Biol Chem 264:5412-5419. inhibition of neurotransmitter release at presynaptic terminal Howell TW, Kramer IM, Gomperts B (1989) Protein phosphorylation The Journal of Neuroscience, December 1994, 14(12) 7703

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