Effects of Protein Kinase C Inhibitors on Ethanol-Induced Contractions in Isolated Rat Aorta

Alcohol, Vol. 18, No. 1, pp. 17–22, 1999 © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0741-8329/99/$–see front matter

PII S0741-8329(98)00062-7

Effects of Protein Kinase C Inhibitors on Ethanol-Induced Contractions in Isolated Rat Aorta TERESA JOVER*, BELLA T. ALTURA*† AND BURTON M. ALTURA*†‡ Departments of *Physiology and †Medicine and ‡The Center for Cardiovascular and Muscle Research, State University of New York, Health Science Center at Brooklyn, Brooklyn, New York Received 21 August 1998; Accepted 30 September 1998 JOVER, T., B. T. ALTURA AND B. M. ALTURA. Effects of protein kinase C inhibitors on ethanol-induced contractions in isolated rat aorta. ALCOHOL 18(1) 17–22, 1999.—The activation of intracellular contractile proteins induces vascular contraction mediated through signal transduction mechanisms. Protein kinase C (PKC) is involved in this signal transduction. The purpose of the present study was designed to investigate the role of PKC on EtOH-, KCl- and phorbol 12, 13dibutyrate (PDBu)-induced contractions in isolated rat aorta through the use of several different PKC inhibitors. Prior exposure to staurosporine inhibited both EtOH- and KCl-induced contractions in a concentration-dependent manner. The EtOHinduced contractions were completely inhibited by staurosporine (5 3 1026 M) but complete inhibition of KCl-induced contractions was not observed. Staurosporine (1027 M) also significantly inhibited the contractile response to single doses of both EtOH and PDBu. Bisindolylmaleimide (1026 M) effectively inhibited contractile responses to both EtOH- and KCl, added cumulatively, and single doses of PDBu. Chelerythrine (1027 M) inhibited maximal EtOH-induced contractions. These results suggest that PKC activation plays an important role in the mechanism(s) involved in the contractile activation of rat aorta smooth muscle by EtOH, KCl and PDBu. However, further work is required to elucidate the precise molecular mechanism. © 1999 Elsevier Science Inc. All rights reserved. Ethanol Protein kinase C Staurosporine Phorbol 12, 13-dibutyrate Rat aorta

Bisindolylmaleimide

Chelerythrine

membrane depolarization, that initiates Ca21 influx through voltage-gated Ca21 channels, leading to a sustained contraction (18). Stimulation of a1-adrenoceptors causes activation of a membrane-bound phospholipase C, which catalyzes the breakdown of phosphatidylinositol-4,5-bisphosphate (PIP2) to release diacylglycerol (DAG) and myo-inositol-1,4,5 trisphosphate (IP3). Formation of IP3 causes release of intracellular Ca21 from intracellular stores in vascular smooth muscle. The released DAG also appears to serve as a second-messenger in vascular smooth muscle contraction by activating PKC in the presence of calcium (25). PKC is translocated to the membrane from the cytosol when it is activated (1). It has been proposed that translocation of PKC may be involved in maintaining the sustained phase of contraction in vascular smooth muscle (31). Support for this theory is derived from studies

SMOOTH muscle contraction is induced by the activation of intracellular contractile proteins mediated through signal transduction from the outside to the inside of cells. Protein kinase C (PKC), a calcium-sensitive phospholipid-dependent protein kinase which is present in high concentrations in vascular smooth muscle (20), has been shown to play an important role in this signal transduction (28,29). Agonist-induced contraction of vascular smooth muscle usually involves both a rapid phasic and a sustained tonic component. These two phases are presumably mediated by different pathways of excitation-contraction coupling. The initial phase of force development has been shown to be associated with an increase in the cytosolic Ca21 concentration via Ca21 entry or release of intracellular Ca21, or both, through Ca21-calmodulin-mediated activation of myosin light chain kinase (13). Typically, KCl activates vascular smooth muscle by

Requests for reprints should be addressed to Professor B.M. Altura, Box 31, SUNY Health Science Center, 450 Clarkson Avenue, Brooklyn, NY 11203. Tel: (718) 270-2194; Fax: (718) 270-3103.

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JOVER, ALTURA AND ALTURA

performed with phorbol esters (11). Phorbol esters, synthetic compounds that can replace DAG in activation of PKC (28), have been shown to contract intact vascular smooth muscle by actions that are variably affected by changes in extracellular Ca21 concentration (19). Moreover, the proteins phosphorylated by phorbol esters are identical to those phosphorylated during the sustained phase of agonist-induced contraction (13). Previous reports also support the notion that PKC activation enhances Ca21 channel activity in smooth muscle cells (26). Phorbol esters stimulate Ca21 channel current in smooth muscle cells from different tissues (24,30). On the other hand, it has been demonstrated that ethanol (EtOH) evokes contractions in peripheral, cerebral and coronary blood vessels from a variety of mammalian species (3– 6,9,14,39). EtOH-induced contractions have been hypothesized to be due to its direct effects on vascular smooth muscle cells. However, the precise mechanism underlying EtOHinduced contraction of vascular smooth muscle is not fully understood (39). It has been suggested that the effects of EtOH may be mediated through its influence on Ca21 availability (6,10) and moreover, that in addition to a need for extracellular Ca21, an intracellular release of Ca21 is needed for ethanol to induce contractions in peripheral, cerebral and coronary vascular smooth muscle (9,39); a reduction in membrane and intracellular free magnesium ions ([Mg21]i) is also thought to be an essential step in EtOH-induced vascular contraction (4,8). The aim of the present study was to investigate the involvement of PKC in the contractile response induced by EtOH in isolated rat aorta; preliminary studies in our lab have suggested that activation of PKC may be essential for vascular-induced contraction (40,41). Previous reports have utilized PKC inhibitors to implicate the involvement of PKC in the contractile response to both phorbol esters and agonists. We examined the possible antagonistic effects of nonspecific (staurosporine) and specific (bisindolylmaleimide I and chelerythrine) PKC inhibitors on EtOH-induced contractions and compared their effects with those obtained on KCl- and phorbol 12,13-dibutyrate (PDBu)-induced contractions in these vascular tissues.

added hypertonically to the organ bath. When the tissues had reached steady-state contraction, the preparations were washed three times with NKRB solution to relax them back to baseline. All contractile responses in each vessel were expressed as a percentage of the preceding contractile response to KCl (50 mM) in that vessel. After another equilibration in NKRB, each experimental protocol was initiated. Effects of Staurosporine, Bisindolylmaleimide I or Chelerythrine on EtOH- or KCl-Induced Contractions Concentration-response curves for EtOH or KCl were obtained by cumulative addition to the organ bath. Either staurosporine, bisindolylmaleimide or chelerythrine, was applied 20 min before the second cumulative concentration-response curve for EtOH or KCl. Effects of Staurosporine or Bisindolylmaleimide I on the Responses to Single Doses of EtOH or Phorbol 12, 13-dibutyrate (PDBu) A single dose of 500 mM EtOH or 1026 M PDBu was employed before, during and after treatment for 20 min with staurosporine or bisindolylmaleimide. Drugs Drugs used in this study were staurosporine (Sigma Chemical Co., St Louis, MO, USA), bisindolylmaleimide hydrochloride I (Sigma), chelerythrine (Sigma), PDBu (Calbiochem-Novabiochem Corporation, La Jolla, CA, USA) and dimethyl sulfoxide (Calbiochem). Staurosporine, chelerythrine and PDBu were dissolved in dimethyl sulfoxide. The other reagents were dissolved in distilled water. The maximum concentration of the solvent, dimethyl sulfoxide, was # 0.1% and had no effect on the contractility of the rat aorta. Statistics

METHOD

Tissue Preparation The experiments were performed on isolated aortae taken from male Wistar rats (250–350 g) (37). The animals were decapitated and the descending aorta was excised. Vascular tissues were cleaned of excess fat and connective tissue and subsequently cut into small segments about 3–4 mm in length. The vascular rings were suspended in 20 ml of NKRB solution of the following composition (in mM): NaCl, 118; KCl, 4.7, KHPO4, 1.2; MgSO4, 1.2; CaCl2, 2.5; glucose, 10; and NaHCO3, 25 (37). Organ baths were maintained at 378C and gassed continuously with a 95% O2–5% CO2 mixture. Resting tensions were maintained at 2 g. Vascular tissues were allowed to equilibrate for 2 h before the experiments were started. The loading tension was adjusted periodically and maintained throughout the equilibration period. During the equilibration period, the bath solution was replaced every 20 min. as a precaution against a build-up of metabolites which could influence contraction. The tissues were attached to force-displacement transducers (Grass Model FT 03) connected to Grass Model 7 polygraphs, and isometric tensions of the vascular smooth muscle preparations were recorded. Subsequently, all tissues were exposed to KCl (50 mM)

All responses were expressed as means 6 SE. Differences between means were analyzed using non-paired t-tests or analysis of variance followed by Newman-Keuls test. Statistical significance was assumed when p , 0.05. RESULTS

Effects of Staurosporine on EtOH- or KCl-Induced Contractions EtOH added cumulatively developed concentration-dependent contractions in rat aorta rings as shown previously (5). Prior exposure of vessels to staurosporine inhibited EtOHinduced contractile responses in a concentration-dependent manner. EtOH-induced contractions were significantly inhibited by staurosporine (1027 M) with a significant decrease in the maximal response and an increase in the median effective concentration (EC50) observed. The contractile response was completely inhibited by staurosporine at a concentration of 5 3 1026 M (Figure 1 and Table 1). KCl-induced contractions were also inhibited, in a concentration-dependent manner, by staurosporine (1027 M), with a significant decrease in the maximal response and an increase in the EC50. The contractile response was not inhibited completely by staurosporine with the concentrations tested (Table 2).

PCK AND EtOH CONTRACTIONS

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FIG. 1. Effects of exposure to staurosporine on EtOH-induced contractions in isolated rat aorta. ED50 values of control and treated tissues are shown in Table 1 (100% maximal response to KCl 5 1502 6 60 mg).

Effects of Chelerythrine on EtOH-Induced Contractions

Effects of Bisindolylmaleimide I on EtOH- or KCl-Induced Contractions Bisindolylmaleimide (5 3 1027 and 1026 M) significantly inhibited KCl- and EtOH-induced contractile responses, respectively. Bisindolylmaleide inhibited KCl-induced contractions far less than did staurosporine (Table 2) consistent with a greater KCl-independence of KCl contractile effects. These data are shown in Tables 1 and 2. TABLE 1 EFFECTS OF PKC INHIBITORS ON ETHANOL-INDUCED CONTRACTIONS IN RAT AORTA

Chelerythrine (1027 M) significantly inhibited the maximal EtOH-induced contractions without producing significant changes in the EC50 (Table 1). Effects of Staurosporine or Bisindolylmaleimide I on the Response to Single Doses of EtOH or PDBu Addition of a single dose of 500 mM EtOH elicited an increase in tension of aorta rings. Prior exposure of aorta segments to staurosporine (1027 M) significantly inhibited the maximal contraction induced by the first and second single doses of 500 mM EtOH (Figure 2). A single dose of 1026 M PDBu produced a slowly devel-

Ethanol Maximal Response (% KCl-Induced Contraction)

Treatment

n

ED50 (mM)

Control Staurosporine 1027 M 5 3 1027 M 1026 M 5 3 1026 M Bisindolylmaleimide 1027 M 5 3 1027 M 1026 M 1025 M Chelerythrine 1027 M 1026 M 1025 M

57

321 6 6

63 6 3

16 16 8 7

380 6 4† 380 6 13† 440 6 0† 2‡

38 6 4† 19 6 2 4 6 1† 0‡

8 8 16 8

393 6 21 270 6 11† 395 6 10 353 6 13

76 6 8 60 6 6 50 6 4* 41 6 5*

4 8 11

368 6 9 331 6 18 317 6 12

35 6 1† 35 6 1* 36 6 5*

Values 5 means 6 S.E.; n 5 numbers of samples. *Significantly different from Control (p , 0.05) †Significantly different from Control (p , 0.01) ‡Highly significant from Control (p , 0.001)

TABLE 2 EFFECTS OF PKC INHIBITORS ON KCl-INDUCED CONTRACTIONS IN RAT AORTA KCl

Treatment

n

Ed50 (mM)

Control Staurosporine 1027 M 5 3 1027 M 1026 M 5 3 1026 M Bisindolylmaleimide 1027 M 5 3 1027 M 1026 M

20

17 6 2

8 8 8 8

24 6 1 69 6 3† 54 6 7† 56 6 7†

8 8 8

19 6 1.7 36 6 7.0* 28 6 5.2

Maximal Response (% KCl-Induced Contraction)

Values 5 means 6 S.E.; n 5 numbers of samples. *Significantly different from Control (p , 0.05) †Significantly different from Control (p , 0.01)

132 6 3 90 6 5† 41 6 3† 31 6 4† 31 6 3† 130 6 7 95 6 8† 102 6 11†

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JOVER, ALTURA AND ALTURA

FIG. 2. Effects of single dose of EtOH (500 mM) on rat aorta before and after incubation with staurosporine (1027 M). Bar on left is tension in mg; horizontal time marker is given in min.

oped and sustained contractile response on aorta rings very similar to that shown by other workers (13,18,19,33,36). Both staurosporine (5 3 1028 M) and bisindolylmaleimide (1026 M) significantly inhibited (e.g., 85–98%) this contractile response (data not shown). DISCUSSION

The present results represent the first demonstration, to our knowledge, that nonselective and selective PKC antagonists inhibit the development of EtOH-induced contraction in isolated rat aorta. Cumulative addition of EtOH resulted in concentrationdependent contractions in rat aorta rings. These results confirm previous reports demonstrating that ethanol evokes contractions in many types of blood vessels including aortae (3– 5,14,39). It has been reported that ethanol directly activates vascular smooth muscle via a modulation of Ca21 metabolism, and that an intracellular release of Ca21 is needed for ethanol to induce contractions (3–5,14,39). Moreover, it has been suggested that ethanol can release intracellular Ca21 that is not related to membrane depolarization and is only partly dependent on extracellular Ca21 (39). Staurosporine (1027 M) was an effective inhibitor of both EtOH-induced contractions and the maximal contraction induced by a single dose of 500 mM EtOH. It has been suggested that staurosporine, which interacts with the ATP-binding site of PKC that shares substantial homology with other protein kinases, may be a non-selective PKC antagonist (32). Nevertheless, both bisindolylmaleimide I and chelerythrine, which are known to inhibit most isozymes of PKC without affecting other protein kinases, and may serve as specific PKC inhibitors (17,35), also antagonized EtOH-induced contractions. These results suggest that the PKC activation is required for EtOH-induced contractile responses of blood vessels. Prior exposure to staurosporine (1027 M) inhibited, greatly, KCl-induced contractions. This result confirms previous reports for this tissue type (12). Since staurosporine is known to be a nonselective PKC antagonist (32), previous studies suggested that staurosporine should not be used as a selective pharmacological tool for the study of the involve-

ment of PKC in smooth muscle contraction (12,33). In the present study, the effect of bisindolylmaleimide I was also tested on the development of KCl-induced contractions. This inhibition was greater than that for EtOH-induced contraction. The development of the contractile response due to these two types of activation involves different pathways of excitation-contraction coupling, suggesting that bisindolylmaleimide I might inhibit these contractions through different mechanisms. In addition, a previous study illustrated the potential of bisindolylmaleimide as a tool to evaluate the involvement of PKC in the contractile function of vascular smooth muscles (35). Therefore, our data might suggest that PKC activation is involved in KCl-induced contractions. In contrast to our results, previous reports using calphostin C, a potent and selective PKC inhibitor, indicated that KClinduced contraction in rat aorta is not due to change of Ca21 sensitivity upon the activation of PKC (21,22,33). The reason for these differences is unknown. However, at least five distinct isoforms of PKC have been identified in vascular smooth muscle (34). These isozymes exhibit different patterns of cellular distribution that are differentially activated (23) and, accordingly, are differentially inhibited. Clearly, this explanation requires further investigation. A single dose of 1026 M PDBu, a phorbol ester, induced a strong contraction in rat aorta rings. Our results thus confirm previous reports in rabbit and rat aorta (33,36). Phorbol esters are known to mimic endogenous DAG in the activation of PKC producing slowly developing and sustained contraction of vascular smooth muscle (13,31). PDBu has been utilized in clarifying the potential function of DAG in the activation of PKC (26). Staurosporine (5 3 1028 M) significantly inhibited PDBuinduced contraction. These results are in agreement with previous reports in the same tissue (16,33). Bisindolylmaleimide (1026 M) also inhibited the contractile response to PDBu, confirming preceding reports that support the view that PKC activation is responsible for the contractile response to PDBu in rat aorta (22,29,36). Since a vascular smooth muscle cell loss of [Mg21]i appears to occur extremely rapidly in response to EtOH (4,8) [and in other cell types (7,15)], and that it has been repeatedly confirmed that Mg21 controls the membrane entry and intracellu-

PCK AND EtOH CONTRACTIONS

21

lar release of Ca21 (2,37,38), it is our belief that the following steps must take place in initiation of EtOH-induced contraction of vascular smooth muscle: (a) a membrane loss of Mg21 (4,8); (b) an entry of some extracellular Ca21 ([Ca21]o as a consequence of [Mg21]i loss (2,38); (c) an increased formation of IP3 and DAG caused by reduction in [Mg21]i and elevation of [Ca21]i (27); (d) a Ca21-mediated activation and translocation of certain PKC isozymes (Ca21-dependent); (e) a PKCactivated enhancement of membrane Ca21 entry and Ca21induced Ca21 release from the sarcoplasmic reticulum (partly as a consequence of [Mg21]i loss and IP3 (2,38); (f) PKC-Ca21 sensitization and phosphorylation of myosin light chain kinase; and (g) activation and interaction of actin with myosin followed by contraction. In conclusion, our observations demonstrate that selective and nonselective PKC inhibitors are effective inhibitors of

EtOH-, KCl- and PDBu-induced contraction. These results appear to suggest that PKC activation plays an important role in the mechanism(s) involved in the contractile function of rat aorta smooth muscle due to these three types of activation, and further work will be required to elucidate this mechanism. A hypothetical signal transduction pathway of EtOHactivation of contraction is suggested based on available experimental data. ACKNOWLEDGEMENTS

The authors thank A. Gebrewold for his excellent technical assistance. T. Jover was a postdoctoral research fellow supported by Conselleria de Educacion y Cienca of the Generalitat Valenciana in the Plan Valenciano de Ciencia y Tecnologia. The authors are indebted to the NIH who gave partial support (AA-08674) to B.M. Altura for the work described herein.

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