Effects of dietary calcium on atrial natriuretic factor release from isolated rat atria

Effects

of Dietary Norman

Calcium on Atria1 Natriuretic From Isolated Rat Atria L.M. Wong,

Factor

Release

Eric F.C. Wong, and David C.K. Hu

Previous studies have shown that acute calcium load causes an increase in circulating plasma atrial natriuretic factor (ANF) levels. The present study was conducted to examine the effect of high dietary calcium on ANF release. Experiments were performed on two groups of male Wistar rats. Hypercalcemic (n = 12) and normocalcemic (n = 12) animals were placed on a high and normal calcium diet, respectively, for 14 days before study. A 24-hour renal clearance was conducted on all animals before super-fusion studies. Clearance results showed that high dietary calcium induced a significant increase in plasma calcium (2.69 2 0.02 v 2.90 t 0.77 mmol/L; P < .Ol). This elevation in plasma calcium was associated with a marked increase in calcium excretion (fractional excretion of calcium, 1.91% + 0.33% v 8.17% 2 0.11%). and was correlated with a significant increase in plasma ANF levels (97 ? 6 Y 167 2 20 pg/mL). We also measured immunoreactive ANF in the atria of hypercalcemic and normocalcemic rats. ANF content and concentration in the atria were lower in hypercalcemic (465 t 36 ng/mg) than in normocalcemic rats (635 * 30 ng/mg). This implies that ANF secretion is stimulated by hypercalcemia. To examine this directly, the right atrium from hypercalcemic and normocalcemic rats was superfused in a modified Langendorff preparation. Spontaneous release of ANF from the isolated right atria of hypercalcemic animals (19 2 0.8 pg/min/mg) was significantly higher (P < .Ol) than from the normocalcemic rats (8.6 2 0.3 pg/min/mg). These results suggest that elevation of plasma ANF levels in hypercalcemia is due to an increase in ANF secretion. Copyright 0 1991 by W.B. Saunders Company

I

ONIZED CALCIUM has been shown to play an important role in the release of a number of hormones.‘.” Early studies by Ruskoaho et al&,’have shown that calcium ionophore Bay K 8644 stimulated atria1 natriuretic factor (ANF) secretion from an isolated perfused rat heart. Thus, it was concluded that calcium is involved in ANF release. Recently, Yamamoto et al’ demonstrated that acute infusion of calcium chloride into anesthetized dogs causes an increase in circulating ANF levels. On the other hand, Fujita et al’ found that when a low dose of calcium chloride was infused into the brachial artery of normal subjects, circulating ANF levels remained unchanged. The effect of dietary calcium on ANF was recently examined by Geiger et al in spontaneously hypertensive rats.’ In this study, an increase in plasma ANF was noted when rats were placed on a high calcium diet for 4 days. We have also examined the effect of an oral calcium load on ANF release in healthy subjects and idiopathic hypercalciuric patients. Our results indicate that ANF release increases in both groups in response to acute oral calcium loads.’ In vitro results were inconsistent. Studies with cultured rat cardiocytes by Matsubara et al”’ found that when calcium channel agonist BAY K 8644 was added to the cultured cells, there was an increase in ANF secretion. On the other hand, the results of Greenwald et al differ.” They reported that when calcium was added to prestretched neonatal myocytes, it caused a reduction in ANF release. Similar results were obtained by DeBold and DeBold with an isolated perfused atrium.”

From the Department of Medicine, UniversityHospital, Universityof British Columbia, Vancomaer, BC. Canada. Supported by a grant from the Medical Research Council of Canada (MAlOOh3) to N.L.M.W. and D.C.K.H. Address reprint requests to Norman L.M. Wang, PhD. Depatiment of Medicine, Universiy Hospital-UBC Site, University of British Columbia, 2211 Wesbrook Mall. Vancouver, BC. Canada V6T 1 W5. Copyright 0 1991 by W B. Saunders Company 0026-049519114005-0006$03.00/0 474

To further our understanding of the role of calcium and ANF secretion, in vivo and in vitro, experiments were conducted in rats to examine the effect of dietary calcium on ANF release. Normal rats, receiving either a normal or a high calcium diet for 14 days, were first examined by 24-hour renal clearances followed by in vitro experiments. The right atria of these animals were isolated and perfused in a modified Langendorff preparation to directly examine the effects of dietary calcium on spontaneous release of ANF by the atria.

METHODS Male Wistar rats ranging between 200 and 225 g in weight were used in our experiments. The animals were housed in metabolic cages and had free access to distilled water and were fed a standard lab chow (Purina Laboratory, St Louis, MO) or a calciumsupplemented diet (37.5 g of CaCOJkg of diet). The normocalcemic animals received the standard lab chow (n = 12) and the hypercalcemic (n = 15) rats were given the calcium-supplemented diet for 14 days. After 14 days. a 24-hour renal clearance was performed and a blood sample was obtained at the end of the 24.hour period. Free-flowing tail vein blood was collected from each rat for electrolyte measurements. Following this clearance, the rats were anesthetized with sodium pentobarbital (25 mg,/kg intraperitoneally). Blood samples were collected from the abdominal aorta for the determination of plasma ANF. The right atria were quickly excised and perfused with a modified Langendorff apparatus as previously described.‘” The atria were superfused at a constant flow rate of 0.5 mL/min at 37°C with Tyrode’s solution (Sigma Chemicals, St Louis, MO), aerated with 95% oxygen, 5O6 carbon dioxide to give a pH 7.4. The perfusion apparatus was placed in a water-jacketed incubator (Napco 4100, National Appliance, Portland. OR). The atria were superfused for 60 minutes to stabilize hormone secretion rate before the experiment started. The experiments were conducted for 100 minutes. The effluent from the perfusion was gathered with a fraction collector (LKB, Broma. Sweden) and placed in storage at -20°C until assay. The levels of ANF in the right atria were determined following the perfusion studies. The atrium was homogenized for 2 minutes in 2 mL of 0.1 mol/L acetic acid and centrifuged for 30 minutes at 30,000 rpm. The supernatant was frozen at -70°C. then thawed Metabolism, Vol40, No 5 (May), 1991: pp 474.477

HYPERCALCEMIA AND ANF RELEASE

475

and centrifuged again at 30,000 rpm for 20 minutes. The pellet was discarded and supernatant was again stored at -70°C until assay. Plasma ANF levels were determined after extraction using a C-18 Sep Pak (Water Associates, Milford, MA). ANF levels were measured by means of radioimmunoassay method as previously described.” ANF antibody was bought from Peninsula Laboratory (Belmont, CA). Synthetic rat (u-ANF (2: 1,000 pg/tube) was used to construct the standard curve. Chloramine-T was used to prepare the “ii-labeled ANF.lJ Free and bound fractions were separated by double-antibody precipitation method. The radioactivity was measured in an LKB 1275 minigamma (Wallet, Finland). The sensitivity of the assay was at least 2 pg of ANF per tube. Sodium was analyzed by flame photometer (Instrumentation Laboratory 943. Milano. Italy). and calcium by atomic absorption spectrophotometry (Jarrel Ash. 850. Pittsburgh, PA). Plasma and urinary creatinine were determined by Abbott VP spectrophotometer (Abbott Laboratory, Dallas, TX). The results were expressed as mean t SEM. Student’s nonpaired t test and ANOVA were used to determine statistical significance of differences. Statistical significance was defined as P < .05, RESULTS Table 1 summarizes the clearance results. Mean body weight and mean blood pressure were similar between the two groups. The glomerular filtration rate as measured by creatinine clearances was the same in both groups. As a result of high dietary calcium intake, plasma calcium was 2.9 2 0.7 mmol/L in hypercalcemic rats as compared to 2.69 5: 0.02 mmol/L in normocalcemic animals. This is further reflected in the urinary excretion of calcium. Fractional excretion of calcium increased to 8.17% t 0.11% in animals receiving high calcium diets, as opposed to 1.91% 2 0.03% in rats receiving regular lab chow. This increase in plasma calcium is associated with a significant elevation in plasma ANF level (167 + 20 pg/mL 1’ 97 ? 6 pg/mL). This is further illustrated in Fig 1. A significant positive correlation (n = IS, r = 38. P < .Ol) is obtained when plasma calcium is plotted against plasma ANF levels with data obtained from the hypercalcemic animals. These results suggest a close relationship between plasma calcium and circulating plasma ANF level. ANF concentration in the atria was significantly lower in hypercalcemic than in normocalcemic rats (635 ? 30 ng/mg v 465 + 36 ng/mg). As

Table 1. Mean Clearance Data Normocalcemla (n = 12)

Body weight (gi MEW (mm Hg) GFR (mUmm) Plasma Na (mmol/L)

328 lr 10 1282 1.88?

Hypercalcemia In = 151

315 i 9

5

120 ?I 7

0.19

1.96 t 0.20

145+- 1

1452

1

FE Na (%)

0.84 k 0.09

0.84 k 0.11

Plasma Ca (mmol/L)

2.69 t_ 0.02

2.90 f 0.07X

FE Ca (%I Plasma ANF (pg/mLj

1.91 k 0.33 97 k 6

a.17 f o.Il*

Tissue ANF (ng/mg)

635 k 30

167 -t 20* 465 2 36*

Abbreviations: MBP, mean blood pressure; GFR, glomerular filtration rate; FE, fractional excretion. *P < .Ol compared with normocalcemic rats.

shown in Fig 2, significant inverse correlations were found between plasma ANF and ANF concentration in the atria (n = 15, r = -.61. P < .Ol). These results suggest that ANF release is chronically stimulated during hypercalcemia. Our results from the in vitro perfused isolated atria confirm this assumption, as illustrated in Fig 3. The isolated right atria obtained from the hypercalcemic rats release more ANF into the perfusate. The difference in ANF secretion between normocalcemic and hypercalcemic persisted for up to 100 minutes, at which time the experiment was terminated. These results suggest that higher circulating plasma ANF level during hypercalcemia is due to increase in ANF release from the atria. DISCUSSION

The results of our present study demonstrated that increased calcium intake in rats led to an increase in plasma ANF. This observation is in accord with the report of Geiger et al’ in spontaneously hypertensive rats. Similar findings were reported when plasma calcium was elevated acutely. Yamamoto et alh showed that intravenous infusion of calcium chloride into anesthetized dogs caused a significant increase in plasma ANF. Patients given an acute oral calcium load also had a higher plasma ANF.” Thus, increases in plasma calcium by dietary means or intravenous administration can caused plasma ANF to increase. Calcium ions are involved in the release of ANF. This is not surprising, since calcium has been considered to be a second messenger in causing the release of several hormones.‘.’ Our in vitro experiments with the superfused right atria showed that the ANF release rate was higher in the hypercalcemic rats. Since these atria were isolated from the body, it suggested that the intrinsic secretory rate was altered in hypercalcemic state. Our tissue measurements support these results. In the present study, we have also determined ANF content in the atria. Hypercalcemia caused a reduction in ANF content in the atria1 tissue while plasma ANF levels were augmented. These observations indirectly suggested that the release of ANF by the atria were altered during hypercalcemia. Recent experiments by Schiebinger’” with isolated rat atria suggested that isoproterenol stimulated ANF secretion is dependent on calcium influx through voltage-dependent channels and on the release of calcium from the sarcoplamic reticulum. All these in vivo and in vitro studies indicate that ANF secretion is a calcium-dependent process. These results are not in agreement with the recent reports of Greenwald et al” and DeBold and DeBold.” The reasons for these discrepancies were not apparent. The experiments of Greenwald et al” were done in cultured myocytes. ANF release was induced by hypo-osmotic shock. Extracellular and intracellular calcium concentrations were altered with EGTA and BAPTA AM. The effect of these compounds on ANF secretion by cultered myocytes is not known. The present study was performed in isolated atria and the experimental designs were different. Alterations in calcium level were achieved by feeding the rats a high calcium diet for 14 days and the increase in plasma calcium was modest. The study by DeBold and DeBold” was also done with an

WONG, WONG, AND HU

476

350 Plasma ANP (pglml) 300

-

250

-

200

-

150100

r =

-

0.88

0.01

p c

50 0 2

I 21

22

23

24

25

26

27

Plasma

28

29

3

31

32

I 33

1 34

I 35

Fig 1. Relationship between plasma calcium and plasma ANF (ANP) obtained from the hypercalcemic animals. A positive correlation was found.

Ca (mM)

dietary calcium intake causes hypercalcemia, which may lead to an increase in intracellular calcium. Whether increase in the absolute intracellular calcium concentration or calcium fluxes from extracellular fluid space into the cell is the major determinant of ANF release is not known at present. Secondary hemodynamic changes associated with hypercalcemia might also influence ANF release. Hypercalcemia may affect blood pressure, heart rate, and right atria1 pressure, which have been shown to affect ANF secretion.‘D Previous studies by Yamamoto et alh concluded that this effect of calcium on ANF is not due to alterations in hemodynamic factors. In addition, the mean arterial pressure measurements recorded in the present study show no difference between the two groups. Thus, changes in blood pressure may not be a factor in ANF release. A number of endocrine abnormalities are related to hypercalcemia, including elevation in epinephrine. norepinephrine,‘7.‘8 and renin activities.“’ Because the reninangiotensin systems have potent aldosterone-stimulating activity and ANF has the opposite effect on aldosterone

isolated atria. EGTA was used to chelate extracellular calcium during the perfusion of the atria. In addition, a small tension was applied during the experiment. In their study, calcium concentrations in the perfusates were abruptly changed to demonstrate the effect of this divalent ion on ANF secretion. In our present study, tension was not applied to the atrium and the amount of calcium in the perfusate remained constant. The increase in plasma calcium was modest in comparison to changes in perfusate calcium used by DeBold and DeBold.” These differences may have contributed to the different results obtained from these experiments. The augmented release of ANF by the atria of hypercalcemic rats seen in the present study may be explained by several mechanisms. One is the direct action of calcium ions in causing ANF release from the atria into the circulation. This is supported by the observation of a direct positive correlation between plasma calcium and plasma ANF. Previous in vitro studies have shown that calcium channel agonists enhance ANF release in cultured cardiocytes’” and in isolated perfused heart,4.5 suggesting that elevation in intracellular calcium causes the release of ANF. Thus, high

Tissue

ANP (ng/mg

800

tissue) 0

r = -0.61 Fig 2.

Correlation

between

plasma ANF

and ANF concentration in the atria of hypercalcemic rats. A significant inverse correlation was found.

1

0 0

25

1 50

1 75

100

125

1

1

I

/

150

175 200

225

250

Plasma

ANP (pg/ml)

p ( 0.01

I _I_._.i_._~_L__i 275

300

325

350

375

4Oij

HYPERCALCEMIA

AND ANF RELEASE

477

ANP Release 3o r

(pg/min/mg)

25 -

‘l’i”“““‘i”“”

0 0

5

10

15 20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95 100

Time (min) Fig 3. Effect of hypercalcemia on spontaneous release of ANF.

-s-

secretion by the adrenal glands, the increase in circulating ANF in the hypercalcemic state may be due to part of a homeostatic mechanism suppressing aldosterone release when renin activity is increased. Increases in adrenergic activity during hypercalcemia may also contribute to the increase in ANF secretion, since in vitro experiments have demonstrated the stimulatory effect of epinephrine on ANF

secretion.4.‘3.“’

Hypercalcemla

+--

Normocalcemia

In summary, we have directly demonstrated that hypercalcemia is associated with an increase in circulating plasma ANF levels. This increase in circulating ANF is due to enhanced secretion of ANF by the atria. ACKNOWLEDGMENT The authors and secretarial

acknowledge the technical assistance assistance of Jessie Bagay.

of Alice Chan

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11. Greenwald JE, Apkon M, Hruska KA, et al: Stretch-induced atropeptin secretion in the isolated rat myocyte and its negative modulation by calcium. J Clin Invest 83:1061-1065, 1989 12. DeBold M, DeBold A: Effect of manipulations of calcium environment on atria1 natiuretic factor release. Am J Physiol 256:H1588-H1594,1989 13. Wong NLM, Wong EFC. Au GH, et al: Effect of u and S adrenergic stimulation on atria1 natriuretic peptide release in vitro. Am J Physiol255:E260-E264,1988 14. Greenwood FC, Hunter WM: The preparation of I”‘labeled human growth hormone of high specific radioactivity. Biochem J 89:114-123, 1963 15. Schiebinger RJ: Calcium, its role in isoproterenol stimulated atrial natriuetic peptide secretion by superfused rat atria. Circ Res 65:600-606. 1989 16. Sato F, Kamoi K, Wakiya Y, et al: Relationship between plasma atrial natriuretic peptide levels and atria1 pressure in man. J Clin Endocrinol Metab 63:823, 1986 17. Marone C. Beretta-Piacoli C. Weidmann P: Acute hypercalcemia hypertension in man: Role of hemodynamics, catecholamines and renin. Kidney Int 20:92-96. 1982 18. Vlachakis ND, Frederics R, Valasquerz M, et al: Sympathetic system function and vascular reactivity in hypercalcemia patients. Hypertension 4:452-458, 1982 19. Walber B, Brenner HR. Guaras H: Hypertension in a patient with hypercalcemia: Captopril and verapamil. Arch Intern Med 142:143-145, 1982 20. Schiebinger RI, Baker MZ, Linden J: Effect of adrenergic and muscarinic cholinergic agonists in atria1 natriuretic peptide secretion by isolated rat atria. J Clin Invest 80:1687-1691, 1987