0013-7227/90/1275-2445$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society

Vol. 127, No. 5 Printed in U.S.A.

Phorbol Esters Enhance Stretch-Induced Atrial Natriuretic Peptide Secretion* HEIKKI RUSKOAHO, OLLI VUOLTEENAHO, AND JUHANI LEPPALUOTO Departments of Pharmacology and Toxicology (H.R.) and Physiology (0. V., J.L.), University of Oulu, SF90220 Oulu 22, Finland

ABSTRACT. Stretching of atrial myocytes stimulates atrial natriuretic peptide (ANP) secretion, but the cellular processes linking mechanical distention to ANP release are unknown. We studied whether or not protein kinase C activation by phorbol ester affects atrial stretch-induced ANP secretion using the modified perfused rat heart preparation that enabled stepwise distension of the right atrium as an experimental model for stretch-stimulated ANP release. The increase in right atrial pressure (2.65 ± 0.13 mm Hg) was accompanied by an increase in the perfusate immunoreactive ANP (IR-ANP) concentration (from 8.3 ± 1.1 ng/5 min to 13.9 ± 2.0 ng/5 min, P < 0.05, n = 14). During stretch, a slight inotropic response was observed, while heart rate and perfusion pressure remained unchanged. Increase in right atrial pressure in the presence of a phorbol ester, 12-0-tetradecanoyl-phorbol-l3-acetate (TPA), known to stimulate protein kinase C activity in heart cells, resulted in a significantly greater increase in the perfusate IR-ANP concentration than after vehicle infusion. The calculated ANP increase corresponding to the 2 mm Hg increase in the right atrial

pressure was 1.52-fold in the control group and 1.84-fold when 10 nM TPA was infused (P < 0.05). Infusion of TPA at a dose of 24 nM further increased the stretch-induced ANP release by causing 2.22-fold (P < 0.01) increase in IR-ANP secretion. As judged by gel filtration chromatography, abnormal release of the large mol wt stored ANP could not account for the secretory response to phorbol ester. Additionally, a phorbol ester analog, 4a-phorbol 12,13-didecanoate, which is incapable of binding to and activating protein kinase C, was inactive as an ANP secretagogue. In contrast, drugs known to increase the concentration of intracellular Ca2+ in myocytes, Bay K8644 (3 and 6 /UM) and forskolin (0.14 nM), significantly inhibited the stretch-stimulated ANP release. This study shows that phorbol ester enhances atrial stretch-stimulated ANP secretion from the isolated perfused heart, suggesting that protein kinase C activity is positively coupled to the stretch-induced ANP release. The results further demonstrate the negative effect of increase in intracellular Ca2+ on stretch-induced ANP release. (Endocrinology 127: 24452455, 1990)

A

TRIAL myocytes synthesize, store, and release atrial natriuretic peptide (ANP), a peptide hormone that is involved in the regulation of blood volume and pressure (1-5). Several physiological manipulations have been identified that acutely increase the circulating levels of ANP, particularly those actions, such as volume expansion, water immersion, and vasoconstrictor substances, that increase atrial wall tension (5, 6). The results obtained using isolated perfused heart, isolated perfused atria, and isolated myocytes are consistent with observations at the level of intact animal atria. In vitro, atrial wall stretch in isolated perfused hearts (7-11) and atria (12-15), as well as osmotic stretch of isolated rat myocytes (16), stimulates the secretion of ANP; however, the precise cellular mechanisms linking mechanical distension to hormonal release are unknown. By analogy to other secretory systems by which the release of proteins are regulated, secretion may involve Received April 9, 1990. Address requests for reprints to: Heikki Ruskoaho, M.D., Department of Pharmacology and Toxicology, University of Oulu, Kajaanintie 52 D, SF-90220 Oulu, Finland. * This study was supported by the Academy of Finland (Helsinki, Finland), Sigrid Juselius Foundation (Helsinki, Finland), and Paulo Foundation (Helsinki, Finland).

a change in the level of a cytoplasmic messenger, such as Ca2+, cAMP, inositol-l,4,5-trisphosphate, and diacylglycerol (17-19). Stretch-activated ion channels have been described in endothelial cells (20), some of which are selective for Ca2+, and stimulation of phosphatidylinositol turnover and formation of inositol-l,4,5-trisphosphate in the isolated perfused rat heart was noted when right atria were dilated (21). We reported (22) that the Ca2+-ionophore A23187, which introduces free Ca2+ into the cell, and phorbol esters, which mimic the action of diacylglycerol by acting directly on protein kinase C, both increase basal ANP secretion in the isolated perfused rat heart. Phorbol ester also increased responsiveness to Ca2+ channel agonists, such as Bay k8644, and to agents that increase cAMP, such as forskolin and membrane-permeable cAMP analogs (23). These results suggested a possible role for calcium-activated protein kinase C in the regulation of basal ANP secretion. The aim of this study was to determine whether or not protein kinase C activation by phorbol ester affects atrial stretch-induced ANP secretion. In addition, the importance of cytosolic Ca2+ as potential intracellular messenger in stretch-stimulated ANP released was studied. The modified perfused rat heart preparation (9) that enabled

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STRETCH-INDUCED ANP RELEASE

2446

the stepwise distension of the right atrium by pressures approximating those found in vivo was used as an experimental model for stretch-stimulated release of ANP from atrial myocytes. The preparation is not subject to neural or hormonal influences. It is, however, exposed to hemodynamic alterations resulting from chemically induced changes in myocardial contractility or in rate of contraction frequency, which appear to be additional stimuli for ANP release (13, 24, 25). Therefore, we made special efforts to keep the cardiac function constant during stretching of right atria. Our present results demonstrate that stretch of the atrial myocytes appears to be positively modulated by protein kinase C activation.

Materials and Methods Animals Male Wistar rats (weighing 250-350 g) from the Department of Pharmacology and Toxicology colony at the University of Oulu were used. The rats were housed in plastic cages in a room with controlled 40% humidity and a temperature of 22 C. A 12h light (0600-1800 h), 12-h dark (1800-0600 h) cycle was maintained. The experimental protocols were approved by the Committee for Animal Experimentation of the University of Oulu. Materials Drugs used in this study were: the phorbol esters, 12-0tetradecanoyl-phorbol-13-acetate (TPA) and 4a-phorbol12,13-didecanoate (4a-PDD) (Sigma Chemical Co., St. Louis, MO), forskolin (Sigma), Bay k8644 (Bayer, Leverkusen, West Germany), synthetic rat ANPi.28 (Sigma), and heparin (Medica, Helsinki, Finland). Other chemicals not mentioned were from Sigma. The phorbol esters were dissolved in dimethylsulfoxide, Bay k8644, and forskolin in ethanol. The final concentration of each solvent was less than 0.03%. Isolated perfused heart preparation The isolated perfused rat heart preparation, which has been described previously (9), was slightly modified. The rats were decapitated 20 min after being anticoagulated with heparin (500 IU/kg body weight, ip). The abdominal cavity was immediately opened, the diaphragm was transected, and lateral incisions were made along both sides of the rib cage. The anterior chest wall was retracted and the heart was cooled with perfusion fluid (4-10 C). The aorta was cannulated superior to the aortic valve, and retrograde perfusion was begun with a modified Krebs-Henseleit bicarbonate buffer, pH 7.40, equilibrated with 95% O2/5% CO2, at 37 C. The final concentrations of the salts in the buffer (millimoles per liter) were: NaCl, 113.8; NaHCO3, 22.0; KC1, 4.7; KH2PO4, 1.2; MgSO4 x 7 H2O, 1.1; CaCl2 x 2 H2O, 2.5; and glucose, 11. Variations in perfusion pressure caused by changes in coronary vascular resistance were recorded on a Grass polygraph (model 7DA, Grass Instruments, Quincey, MA) with a pressure transducer (model MP-15, Micron Instruments, Los Angeles, CA) situated on a side-arm of the aortic cannula. The apex of

Endo • 1990 Vol 127 • No 5

the ventricle was attached to a strain gauge transducer (model FTO3, Grass Instruments) connected to the Grass polygraph at an initial tension of 2 g to record the force of contraction. The output was damped to give a mean contractile force. Heart rate was counted from contractions by the Grass tachograph, and it was increased 15-20% above the spontaneous beating rate by using Harvard Apparatus Stimulator model 345 (Harvard Apparatus, Millis, MA). Pacing also kept the contractile force stable throughout the observation periods. During the equilibration period (60 min) the hearts were perfused by a peristaltic pump (Ismatec SA801, Ismatec Instruments, Zurich, Switzerland) at a flow rate of 7.5 ml/min. In order to confirm the vasoconstrictor effects of infused drugs, the vasculature was dilated by decreasing the perfusion rate to 5 ml/min before experiments began. Right atrial pressure was recorded on a Grass polygraph via a cannula (PE-60) in the inferior vena cava connected to a pressure transducer (model MP-15, Micron Instruments). A glass cannula was inserted into the pulmonary artery for the collection of perfusate. Right atrial pressure could be kept constant at any desired level by adjusting the level of the pulmonary artery cannula tip. Experimental design After a 10 min control period (Fig. 1), a continuous infusion of vehicle or drugs was made via the aortic perfusion cannula using an infusion pump (B. Braun Perfuser ED, Braun Melsungen AG, West Germany) at a rate of 0.5 ml/min for 35 min. Atrial stretch was superimposed for 5 min after 25 min perfusion by elevating the level of the pulmonary artery cannula tip. The coronary venous effluents were collected at 1 or 2 min intervals, placed immediately on dry ice and stored at —20 C until assayed. Control experiments were run with the solvents dimethylsulfoxide and ethanol. Addition of an appropriate concentration of each caused no significant changes in cardiac function or immunoreactive ANP (IR-ANP) in the perfusate. The drug concentrations used were chosen in order to avoid marked effects on basal IR-ANP secretion and hemodynamic variables. TPA was infused in concentrations of 10 and 24 nM, as these concentrations are known to stimulate protein kinase C activity in the isolated perfused rat heart preparation (26); 100 to 160 nM concentrations induce 4- to 5-fold increase in IR-ANP secretion from perfused hearts (22, 23, 27) and cultured myocytes (28) and produce marked negative changes in inotropy and chronotropy (22, 23, 26, 27). The dose of inactive phorbol ester 4a-PDD (29) (44 nM) was designated to be approximately twice that of active phorbol ester. The concentrations of Bay k8644 (3 and 6 fiM) and forskolin (0.14 nM) were chosen because these concentrations in heart cells have been shown to increase intracellular calcium (30,31) and cAMP (32, 33), respectively, and that higher doses produce considerable changes in cardiac function when added to the perfusion fluid (23, 34). The doses of forskolin and Bay k8644 used in this study were 7 to 10 times lower than those previously reported by us to stimulate moderately basal IR-ANP secretion in the perfused rat heart preparation (23). To validate the effects of infused drugs on ANP release and hemodynamic variables in this study, we analyzed the concentration of perfusate IR-ANP and cardiac function immediately before the stretch period and compared them to control values (Fig. 1). Thus, it was possible to examine the effect of com-

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STRETCH-INDUCED ANP RELEASE

2447

EXPERIMENTAL DESIGN

Collection of perfusate

FIG. 1. Experimental protocol. Perfusate for determination of ANP by RIA was collected either at 2 (from —10 min to 0) or at 1 (from 20 to 35 min) min intervals.

Vehicle or drug infusions Stretch

-10

pounds both on basal and stretch-stimulated IR-ANP release in each perfusion experiment. Finally, the effect of higher doses of active phorbol ester on IR-ANP release and hemodynamics was examined by perfusing hearts according to the Langendorff method (spontaneously beating hearts, no load on atria) as described previously (22, 23). In these experiments, vehicle or 24 nM TPA was added to the aortic perfusion cannula at a rate of 0.5 ml/min for 30 min. Assay of IR-ANP in perfusate For the ANP RIA the unextracted perfusate samples were incubated in duplicates of 100 n\ with 100 /il of the middle specific rabbit ANP antiserum in a final dilution of 1:25,000 (35). Synthetic rat ANPi.28, ranging from 0-500 pg/tube, was incubated as standard. The ANP tracer was rat [125I]ANPi.28 from Amersham (Buckinghamshire, U.K.). After incubation for 48 h at 4 C the immuno-complexes were precipitated with sheep antiserum against rabbit 7-globulin in the presence of 500 n\ 1.2 M ammonium sulfate, pH 7, followed by centrifugation at 3000 X g for 40 min. The sensitivity of the assay was 0.8 pg/ tube. The 50% displacement of the standard curve was at 20 pg/tube. The interassay and the intraassay variations were 14% and 5%, respectively. Serial dilutions of perfusate showed parallelism to the synthetic ANP standard. Gel filtration chromatography The molecular form(s) of IR-ANP released from the perfused heart were studied by gel filtration chromatography. The Sephadex G-75 columns (1 X 50 cm) were eluted with 30% acetic acid (3.5 ml/h) at +4 C. The perfusate sample was lyophilized, dissolved in 1 ml 30% acetic acid, and applied to the column. The fractions (1-1.5 ml, depending on the run) were dried in a Speed Vac concentrator, dissolved in the RIA buffer, and analyzed for IR-ANP as described above. The column was calibrated with Blue Dextran (Pharmacia, Vo), purified rat (35), synthetic rat ANP-(l-28) and 125I (Vtot). Data analysis The results are expressed as mean ± SEM. The data were analyzed with two- or one-way analysis of variance (ANOVA).

10

20

25

30

35 min

For the comparison of statistical significance between groups, Student's t test for paired or unpaired data was used. For multiple comparison, one-way ANOVA followed by the Bonferroni t test was used. Differences at the 95% level were considered significant.

Results Basal values of perfusate IR-ANP and hemodynamic variables The mean concentration of IR-ANP in the perfusate before vehicle or drug infusions was 531 ± 28 pg/ml (n = 72). The basal heart rate was 292 ± 2 beats/min, the perfusion pressure was 31 ± 1 mm Hg, the contractile force was 2.1 ± 0.1 g, and the right atrial pressure 1.6 ± 0.1 mm Hg (n = 72). Tables 1 and 2 show the basal values for hemodynamic variables and the concentration of perfusate IR-ANP in each group. The isolated perfused heart preparation was stable for the period of time which was used in these studies. When vehicle was infused without stretch for 35 min, perfusion pressure, heart rate, contractile force (Table 1), and right atrial pressure (Table 2) remained constant; however, a slight decrease (36%, P < 0.05, n = 7) in the concentration of perfusate IR-ANP was noted (Table 2). Previously, a similar decrease in perfusate IR-ANP has been observed by us and others when isolated rat hearts or atria are perfused for 30 min or longer (22, 23, 25, 27, 36). Stretch-induced ANP release Right atrial pressure was varied by manipulation of atrial afterload by means of adjustment of the cannula leading into the pulmonary artery. When the level of pulmonary artery cannula tip was elevated, right atrial pressure increased immediately (Fig. 2). Five minutes of continuous stretch resulted in a marked increase in IRANP detectable in the coronary venous effluent of the perfused rat heart. After reduction of right atrial pressure

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2448

Endo• 1990 Voll27«No5

STRETCH-INDUCED ANP RELEASE

TABLE 1. Effect of atrial stretch and various drugs on hemodynamic variables in the perfused rat heart

Treatment

Heart rate (beats/min)

Perfusion pressure (mm Hg)

Contractile force (g)

Control

Before

Stretch

Control

Before

Stretch

29 ± 1

33 ± 1

33 ± 1

278 ± 6

270 ± 6

Stretch 269 ±7

Before

Vehicle No stretch (n = 7)

2.1

±0.1

2.1 ± 0.1

2.1 ± 0.1

Vehicle Stretch (n = 14)

32 ± 2

36 ± 2

38 ± 1

288 ± 6

287 ± 6

288 ±6

2.1

±0.1

2.1 ± 0.1

2.4 ± 0.1°

4a-PDD, 44 fiM

31 ± 1

41 ± I 6

45 ± 2

290 ± 5

291 ± 6

294 ±6

2.1

±0.1

2.1 ± 0.1

2.3 ± 0.1°

TPA, 10 nM Stretch (n = 10)

31 ± 1

46±5 C

58 ± 8

294 ± 7

284 ± 5

283 ± 7

2.1 ±0.1

2.0 ± 0.1

2.2 ± 0.1

TPA, 24 nM Stretch (n = 9)

32 ± 2

51 ± 2 "

62±3 C

287 ± 6

284 ±7

284 ± 7

2.1 ±0.1

2.1 ± 0.1

2.4 ± 0.1°

Bay, 3 MM Stretch (n = 10)

34 ± 1

35 ± 2

35 ± 2

302 ± 4

300 ± 5

300 ± 4

2.1 ± 0.1

2.2 ± 0.1"

2.4 ± 0.1

Bay, 6 MM Stretch (n = 6)

28 ± 1

31 ± 1

32 ± 1

296 ± 7

296 ± 6

296 ± 6

2.0 ± 0.1

2.6 ± 0.2c

2.8 ± 0.1

Forskolin Stretch (n = 8)

34 ± 4

34 ± 2

36 ± 2

322 ± 14

360 ± 20

374 ± 20

2.0 ± 0.1

2.1 ± 0.2

2.3 ± 0.2

Control

Stretch (n = 7)

After a 10 min control period, vehicle or drugs were added into the perfusion fluid for 35 min (see Fig. 1). Heart rate, contractile force, and perfusion pressure are expressed as means ± SEM and present values at the end of each experimental period. Bay, Bay k8644. The dose of forskolin was 0.14 MM. Values in footnotes (before vs. control or stretch vs. before, Student's t test, paired) are as follows. " P < 0.05. b P< 0.001. c P

RAP (mm Hg)

Endo • 1990 Vol 127 • No 5

VEHICLE NO STRETCH 4-

2-

2T T T T

T

T I. T

llil

T

0

1500-, IR-ANP (pg/ml)

1500-n

1000-

1000FIG. 2. Effect of phorbol esters on the atrial stretch-induced release of IR-ANP in the isolated perfused rat heart. At the time 10 min, as indicated in Fig. 1, vehicle or phorbol esters were added into the perfusion fluid for 35 min. The right atrium was distended for 5 min (hatched area) by elevating the pulmonary artery cannula tip 25 min after the start of vehicle or phorbol ester infusions. RAP, Right atrial pressure. IR-ANP secretion is expressed as picograms per ml perfusate, and values are expressed as mean ± SEM; see Table 1 for number of hearts in each group.

500-

rnTriwrrrrn 20

6-, RAP (mm Hg)

25

30

500-

o-i 20

35

25

30

35

25 30 TIME (min)

35

TPA24nM STRETCH

4a-PDD 44 nM STRETCH

4-

4-

2-

2-

0-

0

1500-, IR-ANP (pg/ml)

1500n

1000-

1000

500-

500-

20

< 0.01) when 24 nM TPA was infused. Further, a linear correlation in response to the stretch was observed between the change in IR-ANP release into the perfusate and the change in right atrial pressure in the presence of phorbol ester infusion (10 nM TPA: r = 0.69, n = 17, P < 0.01; 24 nM TPA: r = 0.66, n = 16, P < 0.01). Heart rate did not change during the distension experiments, but a slight increase in contractile force was noted during stretch when TPA was infused as was also observed when vehicle was added to the perfusion fluid (Table 1). Perfusion pressure increased slowly toward the end of experiment in response to both TPA infusions whereas heart rate, contractile force, and right atrial pressure remained stable (Tables 1 and 2). When ANP values before stretch in the presence of TPA were compared to control values, a small but insignificant decrease in IR-

25 30 TIME (mini

35

20

ANP release into the perfusate was noted (10 nM TPA: 16%; and 24 nM TPA: 7%) (Table 2). To test the specificity of TPA effects via the C-kinase pathway, hearts were perfused with 4«-PDD, a nonactive phorbol ester (29). This phorbol ester did not affect the stretch-induced IR-ANP release; the calculated 2-mm Hg increase in right atrial pressure resulted in a 1.43fold increase in IR-ANP secretion in the presence of 4aPDD infusion, not significantly different from that of the vehicle infusion. When hemodynamic values before distension experiments were compared to control values, heart rate, contractile force, and right atrial pressure remained constant during 4a-PDD infusion, whereas a statistically significant increase in perfusion pressure was noted (Table 1). Basal IR-ANP release decreased about 30% during inactive phorbol ester infusion, as

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STRETCH-INDUCED ANP RELEASE 3.0-,

2451 3.0

n

TPA 24 nM

FIG. 3. The relation between the ratio of IR-ANP release into the perfusate during the stretch to basal release of IRANP and change in right atrial pressure (RAP). Bay, Ca2+-channel agonist Bay k8644. Concentration of 4a-PDD is 44 nM. Results are expressed as mean ± SEM; see Table 1 for number of hearts in each group. *, P < 0.05 and **, P < 0.01 vehicle vs. treatment groups (one-way ANOVA followed by the Bonferroni t test).

o

2.5-

2.5-

TPA 10 nM 2.0-

2.0-

VEHICLE

VEHICLE 1.53

1.5-

4a-PDD

1.5136

^

1.23

^^^£^Z

1.0-1

1.0-1 1

2

1

3

A RAP (mm Hg)

1500-,

. " BAY 3 pM ' • ^ « FORSKOLIN L

2

A RAP (mm Hg)

PERFUSATE IR-ANP

P£RFUSION PRESSURE

— 1000 E

— 120-

60-

FIG. 4. Effect of a phorbol ester (TPA) on the release of IR-ANP into the perfusate and on cardiac function in isolated perfused rat hearts. At the time 10 min, as indicated by arrows, 24 nM TPA (n = 6) or vehicle (n = 10) was added into the perfusion fluid for 30 min. IR-ANP secretion is expressed as picograms per ml perfusate, and each point is the mean value ± SEM.

20

30 400-,

CONTRACTILE FORCE

HEART RATE

i

30

10 TIME (mm)

observed during vehicle administration (Table 2). To exclude the possibility that the active phorbol ester TPA might influence the release of IR-ANP by nonspecific mechanisms or by altering the processing of prohormone, the molecular form of ANP released by stretch and stretch with TPA was analyzed by Sephadex G-75 gel filtration (Fig. 5). As anticipated, stretch caused the release of the low mol wt C-terminal peptide (which comigrated with the synthetic 28 amino acid standard) and little or no detectable prohormone (Fig. 5b). Similarly, rat hearts perfused with TPA released primarily the 28 amino acid ANP-like peptide into perfusate when stretched (Fig. 5d). Bay k8644, forskolin, and stretch-induced ANP release We next evaluated the role of intracellular calcium in stretch-induced ANP release. Several compounds are available that increase the level of Ca2+ in the myocytes

i

1

10

20

30

TIME (mm)

(37), including Bay k8644, which introduces free calcium into the cell (30, 31), and forskolin, a direct activator of adenylate cyclase (32). When these compounds were tested for their abilities to affect ANP release from isolated perfused rat hearts, both decreased the stretchinduced ANP release. If 3 and 6 /uM Bay k8644 was infused, a slight decrease (19-26%) in perfusate IR-ANP levels before the stretch experiment was noted (Table 2), whereas heart rate and perfusion pressure remained unchanged (Table 1). Despite continuous pacing, contractile force dose-dependently increased during Bay k8644 infusions. A significant decrease (26%) in perfusate IRANP levels was also seen during infusion of 0.14 ^M forskolin. Perfusion pressure and contractile force remained constant during forskolin infusion, while heart rate tended to increase, but this change was not statistically significant (Table 1). The increase in right atrial pressure during Bay k8644

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STRETCH-INDUCED ANP RELEASE

2452

A

FIG. 5. Influence of atrial stretch and phorbol ester on the molecular form of ANP released from the isolated perfused rat hearts. Gel Sephadex G-75 gel filtration analyses of perfusates from nonstretched heart without (A) and with (C) the phorbol ester TPA, and stretched heart without (B) and with (D) TPA. The migration of authentic standards is indicated by arrows. The Pro-ANP was obtained from atrial extracts. IR-ANP content in the fractions is expressed as picograms per ml of perfusate per fraction and are therefore directly comparable between the different groups.

C

Pro-ANP

200-

1°

1

Endo • 1990 Vol 127 • No 5

Vtot

1

1

200-

150-

150-

100-

100-

50-

50-

0-

Vo

Pro-ANP

ANP,- 2 8

I

I

1

•-•

n10

20

30 D

30

20

10

Vo Pro-ANP

200-

1

I VNP,. 2 8

I

\

10050-

30

40

0

FRACTION NUMBER

and forskolin infusions resulted in a significantly smaller increase in the perfusate IR-ANP concentration compared to that after the infusion of vehicle alone (Fig. 3b). The calculated ANP increase corresponding to the 2-mm Hg increase in the right atrial pressure was 1.36-fold and 1.18-fold in the presence of 3 M and 6 /xM Bay k8644, respectively, and 1.23-fold when 0.14 ^M forskolin was infused. Heart rate and perfusion pressure did not change during the distension experiments, whereas a similar increase in the force of contraction was noted both after Bay k8644 and forskolin infusions as observed in vehicle groups (Table 1).

Discussion Stretching of atrial myocytes in vitro stimulates ANP secretion, but the biochemical cellular processes involved in linking mechanical distension to ANP release are unknown. In this study, we used a modification of the perfused rat heart preparation which permitted distension of the right atrium (9) to examine the mechanisms involved in atrial stretch-induced ANP release. A mean increase in right atrial pressure of 2 mm Hg produced by elevation of the pulmonary artery cannula tip resulted in a 52% increase in the rate of ANP release into the perfusate. These data are in good agreement with our previous report in which right atrial pressure was increased between 0.4 and 4.5 mm Hg under similar experimental conditions, and IR-ANP release into the perfusate exhibited a linear correlation between the change in atrial pressure (9). The present results further indicate that a phorbol ester, TPA, which enters cells and activates protein kinase C (19), dose dependency augmented stretch-induced ANP release by shifting the ANP vs. right atrial pressure curve to the left, i.e., for a given increase in the degree of stretch in the presence of

I

^

020

40 Vt0,

1

A -

150-

10

Vtot

•1

10

A

20

30

40

(ml)

phorbol ester more ANP was released. Thus, the activity of the protein kinase C appears to positively regulate stretch-induced ANP release. Protein kinase C is localized in both membrane and cytosolic fractions in the heart (38, 39), and its activity appears to be higher in atria than in ventricles (40). Both in isolated perfused rat hearts (22, 23, 27) and isolated cultured myocytes (28, 41, 42) ANP secretion has been shown to be stimulated by TPA. In perfused, spontaneously beating rat hearts, TPA (15-160 nM) produced a dose-dependent increase in IR-ANP secretion (22, 23, 27), and TPA combined with Ca2+ ionophore A23187 (22), Ca2+ channel agonist Bay k8644 (23), or a axagonist, methoxamine (27) produced a synergistic effect. In primary cultures of cardiac atrial myocytes, a system that allows the secretory response in the absence of neural, hormonal, or hemodynamic stimuli to be studied, TPA caused maximal ANP release into the incubation medium at a concentration of about 100 nM (28). In adult isolated cells TPA stimulated ANP secretion without requiring Ca2+ influx or ryanodine-inhibitable release of Ca2+ from sarcoplasmic reticulum, although the absolute magnitude of TPA-stimulated ANP concentrations was larger for the contracting cells (42). In this study, we confirmed our initial observations of the effects of TPA on basal ANP secretion and demonstrated that 10-24 nM TPA, known at these concentrations (10-100 nM) to activate protein kinase C in this experimental model (26), augments atrial stretch-stimulated ANP release. The linearity between the right atrial pressure and ANP release, observed previously (9), was also maintained in the presence of phorbol ester infusions. Additionally, our present results demonstrate that a phorbol ester analog, 4a-PDD, which is incapable of binding to and activating protein kinase C (29), was

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STRETCH-INDUCED ANP RELEASE

also inactive as an ANP secretagogue. Finally, as judged by mol wt sizing, abnormal release of the large mol wt stored ANP could not account for the secretory response to phorbol ester under these experimental conditions. Thus, the results from this and previous studies demonstrate that calcium-activated protein kinase C appears to play an important role in the regulation of basal and stretch-induced ANP release (Table 3). It remains to be determined whether atrial wall stretch alone or factors liberated in response to distension are responsible for the activation of protein kinase C and ANP secretion. «i-Adrenergic (43), angiotensin II (44), and vasopressin-1 (45) receptors are coupled to phosphoinositide hydrolysis in cultured cardiocytes and when stimulated probably cause the diacylglycerol-mediated stimulation of protein kinase C (17, 19). Since TPAmediated protein kinase C activation appears to stimulate both basal and stretch-stimulated ANP release, hormones and neurotransmitters that activate phosphoinositide hydrolysis in heart cells would be expected to influence stretch-mediated ANP secretion. The fact that vasopressin, angiotensin II, and adrenaline augment the atrial stretch-induced ANP release in vivo supports this (46, 47). Further, «i-adrenergic agonists and vasopressin have been shown to stimulate basal release of ANP in different in vitro models (22, 27, 28, 41, 48-54). In contrast, others have reported that these hormones and neurotransmitters did not influence ANP release in vitro (55, 56). Moreover, muscarinic cholinergic agonists, which also stimulate phosphatidylinositol hydrolysis in heart cells (43), have been reported to increase (22, 41, 50) or inhibit (51) ANP secretion. Thus, the activation of phosphatidylinositol hydrolysis alone is not sufficient to stimulate IR-ANP secretion from atrial myocytes. The failure of all hormones and neurotransmitters that activate phosphoinositide hydrolysis in heart cells to stimulate ANP secretion may be partially explained by the different effects of phorbol esters and other stimuli on protein kinase C activity, which have been described recently in cultured cardiocytes (57), while still acting via the same intracellular second messenger. To further examine the biochemical cellular mechaTABLE 3. Effect of various drugs mimicking the effect of a particular second messenger on ANP secretion from isolated perfused rat hearts Effect on ANP release Second messenger

Drug TPA 4a-PDDc Bay k8644 Forskolin

Unstretched atria"

Stretched atria6

DAG Ca2+ cAMP

|, Increase; J, decrease; • >, no effect; DAG, diacylglycerol. " From Refs. 22 and 23. 6 From present study. c Inactive phorbol ester.

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nisms involved in stretch-stimulated ANP release, we evaluated the importance of cytosolic Ca2+ as possible intracellular messengers. In the present study, Bay k8644, a compound that increases the concentration of intracellular Ca2+ (30, 31), dose dependency inhibited the stretch-stimulated ANP release. This inhibition was also observed when distension experiments were done during infusion of forskolin, which stimulates adenylate cyclase and elevates intracellular cAMP concentration (32, 33), thereby causing an increased calcium influx into the cell (37). The concentrations of Bay k8644 and forskolin used were similar to those shown to be effective in heart cells (31, 33). These data suggest that Ca2+ is not the intracellular messenger for stretch-stimulated ANP release. In contrast, increasing cytosolic Ca2+ appears to be a negative modulator of the release process. Previously, Ito et al. (11) have reported that ANF release was partially suppressed in the calcium-depleted hearts, but the calcium-deficient Krebs medium also abolished the contractility, and that the absence of calcium increased total ANF release. In accordance with the present study, others have shown that Ca2+ or cAMP may negatively modulate secretion of ANP from the isolated perfused atria and isolated atrial myocytes (15,16). Thus, it is apparent that increased entry of extracellular calcium is not necessary for cellular release of ANP by stretch. Interestingly, previous studies using different experimental models of ANP release have shown that drugs which increase intracellular concentration of Ca2+ such as the calcium channel agonist Bay k8644 and the Ca2+ ionophore A23187 stimulate basal ANP release (22, 23, 36,41,42,58). Several recent reports illustrate that atrial contraction frequency and the force against which the cells contract may explain this discrepancy (25, 28, 42, 59, 60, 61). Bay k8644 has been reported to increase ANP secretion in spontaneously beating or electrically paced perfused hearts and atria and in contracting isolated myocytes (23, 42, 59), suggesting that enchanced calcium influx is able to augment ANP secretion. In contrast, when isolated cells which do not contract or arrested atria and hearts (inhibited by chemical compounds) are examined, Bay k8644 has no effect (42, 59). In fact, it has been recently reported that that forskolin lowers ANP secretion in nonbeating cultured atrial cardiocytes (61). Taken together, the results of this and other studies show that secretion of ANP in vitro in response to changes in intracellular calcium and cAMP appears to depend on both the contraction frequency and atrial stretch. When atrial myocytes beat spontaneously, drugs known to increase the concentration of intracellular Ca2+ moderately increase IR-ANP release. In contrast, when atria are stretched, increasing cytosolic Ca2+ appears to inhibit the release process. Thus, these data suggest that,

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STRETCH-INDUCED ANP RELEASE

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during diastole when atria are stretched and the intracellular level of Ca2+ decreases, it is the activation of protein kinase C which may contribute to stretch-induced ANP release. Hintze et al (62) in support of this demonstrated that ANP secretion best correlates with atrial wall stress measured during passive diastolic, not systolic, atrial filling. The effects of phorbol ester and atrial stretch are essentially independent of contraction frequency and predetermined stretch, unlike Ca2+. Finally, it is not yet clear how activation of protein kinase C and stretch could lead to release of ANP from atrial cells. One possibility is that they may produce phosphorylation of certain cellular proteins involved in secretory process of ANP from atrial storage granules. In isolated rat cardiocytes, TPA enhances the phosphorylation of troponin I (63). In vascular smooth muscle, arterial stretching have been shown to increase myosin light chain phosphorylation in proportion to the extent of stretch (64). Further, protein kinase C activation by TPA augments stretch-induced tone in vascular smooth muscle (65), as it enhances atrial stretch-mediated ANP release as shown in this study. Thus, experiments in vitro comparing stretch and protein kinase C activation should provide very interesting information on the mechanisms of stretch-induced ANP secretion. In conclusion, when spontaneously beating rat hearts are perfused by the Langendorff method, ANP is released into the perfusate. This release can be enhanced physiologically by atrial stretch. Our present results demonstrate that stimulation of ANP release in vitro by stretch is positively modulated by phorbol esters suggesting that the calcium-activated protein kinase C plays an important role in the regulation of both basal and stretchstimulated ANP release. In contrast, drugs that increase the intracellular concentration of calcium inhibited ANP secretion showing that the increased entry of extracellular calcium is not necessary for atrial stretch-stimulated ANP release.

Acknowledgments We thank Ms. Eira Kerala and Mrs. Tuula Raisanen for expert technical assistance.

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