Interaction between stretch and hormonally atria1 natriuretic peptide secretion RICK

J. SCHIEBINGER

AND

KELLY

stimulated

M. GREENING

Department of Medicine, Wayne State University, Detroit 48201; Allen Park, Michigan 48101 Schiebinger, Rick J., and Kelly M. Greening. Interaction between stretch and hormonally stimulated atria1 natriuretic peptide secretion.Am. J. Physiol. 262 (Heart Circ. Physiol. 31): H78H83, 1992.-Atria1 stretch and paracrine hormones stimulate atria1 natriuretic peptide (ANP) secretion. The potential interplay between atria1 stretch and paracrine hormoneswasexamined.Isolated superfusedrat left atria paced at 4 Hz were used for study. The effects of 0, 0.5, and 1.5 g settings of initial tension on the ANP secretory responseto 1 PM norepinephrineand 10 nM endothelin were examined. The peak ANP secretory responsesexpressedas a percent of baseline for each of the tension settings were 109 k 8, 132 t 6, and 171& 10%for norepinephrineand 285 t 15,294 t 12,and 368 t 19 for endothelin, respectively. The effects of 0.5 PM norepinephrine, 1 nM endothelin, and 100 nM vasopressin on stretch-stimulated secretion were examined. Norepinephrine and endothelin increasedANP secretion 144 t 16 and 136 t 2% above baseline,respectively. Vasopressindid not increase ANP secretion.Norepinephrine and vasopressindid not significantly influence the ANP secretory responseto stretch. In contrast, endothelin increasedthe responseto stretch by 33% (P < 0.035). We conclude 1) the greater the degreeof atria1 stretch, the greater is the responseto norepinephrine and endothelin; 2) endothelin enhancesthe secretory responseto stretch; and 3) norepinephrine and vasopressindo not affect stretch-stimulated release.Theseresultspredict a greater ANP secretory responseto hormonal stimulation in vivo in volumeexpandedstates.

endothelin; norepinephrine;vasopressin;atria; rat; in vitro NOREPINEPHRINE AND ENDOTHELIN increase atria1 natriuretic peptide (ANP) secretion. Each has a direct atria1 effect to enhance ANP secretion in vitro (8, 10, 23, 24). Thus endogenous sympathetic tone or endothelial cell release of endothelin may modulate ANP secretion in vivo. Atria1 distension or stretch also increases ANP secretion both in vitro and in vivo (1, 7, 13, 14, 25). It is possible that the degree of atria1 wall tension (stretch) may influence the ANP secretory response to hormonal stimuli. Contrariwise, the level of sympathetic tone or amount of endothelin released by endothelial cells may influence the secretory response to stretch. In this study we examine the potential interplay between hormonal stimuli of ANP release and atria1 stretch. Vasopressin, when administered in vivo, increases plasma ANP (15). However, as a pressor agent, vasopressin also increases blood pressure, resulting in a rise in atria1 pressures. Thus it is unclear whether the vasopressin-induced rise in plasma ANP is reflective of a direct atria1 effect or mediated by an increase in atria1 pressure. There are conflicting reports regarding the direct effect of vasopressin on ANP secretion in vitro (4, 12, 30). In this study we examine the direct effect of vasopressin on ANP secretion.

and Veterans Affairs Medical Center,

Lastly, in a recent in vivo study, vasopressin enhanced the rise in plasma ANP in response to volume expansion in the pithed rat (20). The speculation was made that vasopressin may enhance the ANP secretory response to volume expansion by a direct atria1 effect. In this study we determine whether vasopressin directly influences the ANP secretory response to stretch. METHODS

Materials werepurchasedfrom the following sources:human endothelin, rat cu-ANP,and ANP antibody werefrom Peninsula Laboratories (Belmont, CA); medium 199 was from GIBCO (Grand Island, NY); norepinephrine wasfrom Sigma(St. Louis, MO); vasopressinfrom Bachem (Torrance, CA); and goat antibody to rabbit gamma globulin was from Calbiochem (San Diego, CA). Atria1 superfusionexperimentswereperformed aspreviously described (26). Isolated, superfusedrat left atria from female Sprague-Dawleyrats weighing 225-250 g were used for study. To determine the effect of resting tension on stimulated secretion (seeFigs. 1 and 2), rat left atria were mounted with initial tension settings of 0,0.5, or 1.5 g and pacedat 1 Hz. Atria with 0 g tension were slightly stretched to determine the threshold for pacing followed by removal of all tension. No adjustments in tension were madeafter the initial setting. After 30 min the pacing frequency was increased to 4 Hz. At 85 min after mounting, baseline sample collection was started (time 0 in Figs. 1, 2, and 5). Norepinephrine experiments wereperformed with 100PM ascorbicacid to decreaseoxidation. To determine the effect of stimuli on stretch-stimulated ANP secretion (see Figs. 3,4, and 6), left atria were initially stretched to 0.5 g after mounting. Atria were paced as describedabove. Superfusion with vasopressin,norepinephrine, or endothelin was initiated 100 min following atria1 mounting, which was 30 min before stretching the atria to 1.5 g. ANP measurementswere performed on timed fractions of the superfusateby radioimmunoassayas previously described(26). To demonstratethe biological activity of vasopressin,rat thoracic aorta rings, 2 mm in length, were mounted in the atria1 chambers.The rings were stretched to 1.75g and were allowed to equilibrate over 30 min before the addition of vasopressin.The responseto vasopressin was monitored for 20 min. Statistical analysis was performed by paired or unpaired t test or repeated measuresanalysis of variance using StatView II (Abacus Concepts,Berkeley, CA). Resultsare meanst SE. They are expressedas a percent of baseline,which is defined as the mean of seven samplesobtained immediately before experimental manipulation. The results of the stretch experiments are expressedas a percent of the net responserelative to baseline(stretch responseminusmeanof 3 samplesobtained immediately before stretch divided by the mean of 7 baseline samplesobtained before addition of the hormonal stimulus). RESULTS

The influence of resting tension on the secretory response to submaximal stimulating concentrations of nor-

H78

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INTERACTION

OF

STRETCH

AND

epinephrine (1 PM) and endothelin (10 nM) was examined. The immunoreactive ANP secretory response to norepinephrine was dramatically affected by the initial level of tension placed on the atria (Fig. 1). The ANP secretory response to norepinephrine rose with each incremental increase in initial tension. Before the addition of norepinephrine, resting tension was higher in those atria initially stretched to 1.5 g than in the 0.5-g group (Table 1). The reason that resting tension was lower than that initially placed on the atria is due to the inherent property of continuous relaxation following stretch. After the addition of norepinephrine, resting tension fell insignificantly to 0.53 t 0.04 g in the 1.5-g group and remained unchanged in the 0.5-g group. The increase in developed tension in response to norepinephrine was also greater in atria stretched to 1.5 g (Table 1). Figure 2 illustrates the effect of the resting tension level on endothelin-stimulated ANP secretion. The influence of resting tension on endothelin-stimulated secretion differed from that of norepinephrine. The secretory responses at 0 g initial tension and 0.5 g were similar. However, the response at 1.5 g was greater than that of 0 or 0.5 g. As in the norepinephrine experiments, resting Nonpi.nephrina

HORMONES

ON

ANP

H79

SECRETION

Endothelin

10 nM I

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T

150

= 0 g tension = 0.5 g tension A = 1.5 g tension

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45

60

TIME (min) Fig. 2. Effect of resting tension on endothelin-stimulated immunoreactive ANP secretion. Superfused rat left atria were paced at 4 Hz. Initial levels of tension applied to atria were 0 (n = 8), 0.5 (n = 7), and 1.5 g (n = 8). Beginning at 15 min, atria were continuously superfused with 10 nM endothelin. Results are expressed as in Fig. 1. The 1.5-g curve is significantly different from other 2 curves (P c 0.001). Basal ANP secretion was 213 t 26 (0), 347 t 55 (o), and 295 t 54 pg/ml (A). Differences in basal secretion were not significantly different.

Table

2. Effect of initial tension settings on parameters of atria1 performance in response to 10 nM endothelin Initial 0.5

R’C g

= 0 g tension 0 = 0.5 g tension A = 1.5 g tension

0

1

1

I

I

J

0

15

30

45

60

TIME (min) Fig. 1. Effect of resting tension on norepinephrine-stimulated immunoreactive atria1 natriuretic peptide (ANP) secretion. Superfused rat left atria were paced at 4 Hz. Initial levels of tension applied to atria were 0, 0.5, and 1.5 g. Beginning at 15 min, atria were continuously superfused with 1 PM norepinephrine. Results are expressed as a percent of baseline defined as mean of 7 measurements obtained between 0 and 15 min. Three curves (n = 6 atria/curve) are significantly different (P < 0.01). Basal ANP secretion was 279 2 39 (0), 368 t 38 (o), and 299 k 35 pg/ml (A). Differences in basal secretion were not significantly different.

Table

1. Effect of initial tension settings on parameters of atria1 performance in responseto 1 FM norepinephrine Initial 0.5

w g

DT (before), g DT (after), g ADT, g

Tension,

0.18kO.02

DT (before), g 0.07*0.01 DT (after), g 0.12+0.02t 0.05~0.01 ADT, g Values are means t SE. Abbreviations in Table 1. * P c 0.014 compared with with before endothelin addition.

g

1.5

0.13zkO.02 0.05~0.01

0.54~0.04" 0.11t0.01*

0.12&0.02j0.07~0.01

0.27+0.03*-f0.16t0.03*

Values are means t SE. RT, resting tension measurement before addition of norepinephrine; DT, developed tension measurement before or after addition of norepinephrine; ADT, net increase in DT after addition of norepinephrine. * P < 0.007 compared with 0.5 g. t P c 0.002 compared with before norepinephrine addition.

Tension,

g 1.5

0.50&0.02* O.llt0.01* 0.24+0.03*t

0.13&0.02* are the same as those used 0.5 g. “r P c 0.009 compared

tension was higher in those atria initially stretched to 1.5 g than the 0.5-g group (Table 2). Resting tension remained unchanged in both groups following the addition of endothelin. Again, the increase in developed tension in response to endothelin was greater in atria stretched to 1.5 g. The effect of norepinephrine and endothelin on stretch-stimulated ANP secretion is illustrated in Figs. 3 and 4. Atria were superfused with near threshold concentrations of norepinephrine (0.5 PM) or endothelin (1 nM) for 30 min before stretching, which raised ANP secretion by 144 t 16 and 136 t 2% above baseline and raised developed tension by 2.1 t 0.2- and 1.8 t O.l-fold, respectively. Stretching the atria to 1.5 g produced an immediate rise in ANP secretion followed by a rapid fall. Superfusion with norepinephrine (Fig. 3) did not significantly increase the peak ANP secretory response to stretch. A more rapid fall in ANP secretion following the peak response that occurred in atria superfused with norepinephrine, however, did not reach statistical significance (P = 0.20). A fall in ANP secretion was also seen in control nonstretched atria superfused with norepinephrine. A fall in ANP secretion by control atria was not observed in Figs. 4 and 6. Thus the greater fall in ANP secretion following stretch by atria superfused with norepinephrine appears to be due to a characteristic rise and fall in ANP secretion following stimulation with

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H80

INTERACTION

OF STRETCH

AND HORMONES

norepinephrine at these concentrations. Time 0 in Fig. 3 is equivalent to 25 min in Fig. 1. In contrast, endothelin significantly (P c 0.035) enhanced the response to stretch (Fig. 4). The peak response was raised by 33%. Resting tension measurements obtained before stretching the atria were similar in atria superfused with or without each hormone (Table 3). Superfusion with 100 nM vasopressin failed to increase = stretch alone = stretch + norepinephrine = norepinephrine alone

ON ANP SECRETION

ANP secretion (Fig. 5). Likewise, 100 nM vasopressin failed to significantly increase the peak response to stretch (P = 0.24; Fig. 6). The biological activity of vasopressin was demonstrated by superfusion of rat aortic rings with the same buffer containing 100 nM vasopressin. Vasopressin increased aortic ring tension from 1.14 t 0.08 to 1.74 t 0.12 g (P < 0.001, n = 6), whereas tension fell from 1.26 t 0.08 to 1.24 t 0.08 g in rings superfused with buffer alone (n = 6). DISCUSSION

The level of resting tension influences the ANP secretory response to submaximal stimulating concentrations of norepinephrine and endothelin. The-higher the level of resting tension, the greater the secretory response. The mechanism responsible for this phenomenon is not known. However, it may be due to a greater rise in the cytosolic calcium concentration at higher levels of resting tension (31) in response to hormonal stimulation. The enhanced secretory response to hormonal stimuli at

-40

0

10

20

30

40

50 150

TIME (min) Fig. 3. Effect of norepinephrine on stretch-stimulated immunoreactive ANP secretion. Superfused rat left atria (n = 5 atria/curve) were paced at 4 Hz. Initial tension was set at 0.5 g. Atria were continuously superfused with 0.5 PM norepinephrine or buffer for 30 min before stretching. At 5 min, atria were stretched to 1.5 g or were left alone. Results are expressed as a percent of the net response to stretch relative to mean of 7 baseline samples obtained before superfusion with norepinephrine. 80 -

0 = stretch alone 0 = stretch + endothelin A = endothelin alone

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