Endogenous digitalis-like substance in pig left ventricle

Life Sciences, Vol. 39, pp. 2483-2492 Printed in the U.S.A.

ENDOGENOUS DIGITALIS-LIKE

Pergamon Journals

SUBSTANCE IN PIG LEFT VENTRICLE

J.C. Khatter*, M. Agbanyo and R.J. Hoeschen Section of Cardiology, Department of Medicine and Pharmacology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada R3E 0Z3 (Received in final form September 17, 1986) Summary A crude fraction was isolated from pig heart left ventricle (150g) homogenates after extraction of lipids, chromatographic separation and desalting. The extract contained an ionic content of 0.21, 0.27, 0.33 and l. TmM respectively for Mg 2+, Ca 2+, K +, and Na +. The albumin extract, used as a reference control, contained an ionic content of 0.88 and 2.1mM respectively for K + and Na + and negligible amounts of Mg 2+ and Ca 2+. The isolated fraction exhibited digitalis-like properties in i) the inhibition of sareolemmal Na +, K+-ATPase in a dose dependent manner, 2) the displacement of [3H]ouabain binding from membrane receptor sites and 3) produced +ve inotropic response in isolated perfused heart in a dose dependent manner. The albumin extract tested in the same manner showed no digitalis-like properties. The ventricular fraction was unable to displace (-) 3HDHA binding from membrane sites and its inotropic action was not blocked by propranolol. The data suggests that the fraction isolated from pig heart left ventricle contains a substance which has some properties like digitalis. It has been known since the early experiments of Sidney Ringer in 1885 (i) that serum has a stimulant action upon isolated heart. Clark in 1913 (2) confirmed these findings and suggested that the active substance in alcoholic extract of serum was a soap, which exerted its action by effects on the fixation of Ca 2+ on the surface of heart muscle. Later some of the sapotoxins, such as bufotalin secreted by toad skin was found to have digitalis-like action upon the heart and Faust in 1921 (3) proposed that perhaps animals produced their own "digitalis" even under normal conditions. More recently, with the availability of high specific activity radioligands and the advent of highly sensitive radioceptor assay, the investigations of endogenous digitalis-like substances (DLS) have drawn some attention (4). LaBella and his group (5,6) have recently screened a variety of compounds (including naturally occurring steroids and their derivatives) as possible candidates for digitalis-like substance (DLS). A variety of other reports indicate immunoreactive digitalislike activity in plasma of volume expanded dogs (7), human volunteers (8) and in serum of rats with experimental cardiac overload (9). A recent report suggests that the serum of patients with essential hypertension contains a substance which influences Na +, K + transport and has ouabain-like activity (i0). Digitalis-like immunoreactivity has also been reported in neonatal serum and placental extracts (ii). In addition several reports have provided some evidences of the existence of DLS in brain (12,13), rat and guinea pig cardiac tissues (14,15) and rat adrenal glands (16). In this report we wish to document characteristic properties of DLS fraction extracted from pig left ventricular cardiac tissue. Copyright

0024-3205/86 $3.00 + .00 (c) 1986 Pergamon ,Journals Ltd.

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Methods Pig heart left ventricular tissue (150 g) was trimmed of fat and connective tissue. The tissue was then homogenized in ice-cold acetone: 0.I M HCI (60:40; i0 ml/g tissue) using polytron homogenizer. The homogenate was stirred on ice for two hours and centrifuged at 27,000 x g for 30 minutes. The supernatant (supernate I) from this centrifugation was saved. The pellet resuspended in acetone: 0 . 0 1 M HCI (60:40; 5ml/g original tissue weight), homogenized, stirred for 30 minutes on ice and centrifuged at 27,000 x g for 30 minutes at 4°C. The supernatant from the second centrifugation was combined with supernatant I and frozen overnight at -50°C, thawed and centrifuged at 27,000 x g for 30 minutes at 4°C. The supernatant thus obtained was concentrated under reduced pressure at 35°C to approximately 200 ml. The concentrate was centrifuged at 27,000 x g for 30 minutes. To the supernate resulting from this centrifugation, 50 ml of methanol was added and the resulting precipitate removed by centrifugation at 500 x g for 10 minutes at 4°C. The supernatant obtained was then extracted with 4 x 150 ml diethyl ether. The aqueous phase was then concentrated under reduced pressure at 35°C to approximately 10 ml and applied to Sephadex G-25 column. Column Chromatography Sephadex G-25-80 (fine bead) was obtained from Sigma Chemical Co. and swollen overnight in water at room temperature. A glass column (85cm x 5.3cm 2) was filled with the gel and was allowed to settle under gravity. The column was then allowed to equilibrate overnight with 50 mM Tris (pH 7.4). The void volume (175 ml) was determined with Dextran blue (exclusion limit 2 x 106). The crude extract (i0 ml), as obtained above, was applied and the column was eluted with 50 mM Tris-HCl buffer (pH 7.4). The flow rate was adjusted to 1 ml/min and 1.5 ml fractions were collected after accounting for the void volume. The collected fractions were tested for an inotropic response using Langendorf preparation of isolated perfused guinea pig heart. The active fractions, (55-75) were pooled, concentrated under reduced pressure at 35°C to approximately 5 ml. The pH of the concentrate was then adjusted to pH 7.4, with i M Trizma base and the resulting precipitated protein~ removed by centrifugation at 27,000 x g for 15 minutes. The supernate was then brought to pH 4.0 with 12% TCA and the resulting precipitates removed by centrifugation at 27,000 x g for 15 minutes. The pH of the resulting supernate was adjusted to pH 7.0 with 1 M Trizma base, evaporated to dryness, and reconstituted in 5 ml distilled deionized water. For reference control experiments, 150 gm bovine albumin (FR V) replaced the cardiac tissue in the above extraction procedure. Membrane ATPase and 3H-ouabain bindin$ Male guinea pigs were killed by cervical dislocation and their heart removed. The membrane fraction was isolated and the activities of Na +, K+-ATPase and [3H]-ouabain binding were measured by the methods described by Khatter and Hoeschen (17). The protein concentration was determined by the method of Lowry et al (18). The data were analyzed by student's t test and linear regression analysis. Studies on isolated perfused hearts Isolated hearts from guinea pigs of either sex were used for these studies. Using Langendorff preparation, the hearts were perfused at 32°C with Krebs-Henseleit solution (pH 7.4). The method of perfusion was the same as

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described previously (19). Hearts were paced at 180 beats/minute using SRI stimulator No. 6030. The effects of DLS and the other related studies were carried out by injections through the side channel just above the heart cannula In this manner the time required by the drug to reach the heart was negligible and the injected drug was continuously washed by the perfusate. Binding of (-) 3H-dihydroalprenolol

[(-) 3H-DHA]

to myocardial membranes

Effects of DLS was investigated on the (-) 3H-DHA binding to adrenoceptors in guinea pig myocardial membranes by the method described by Karliner et al (20). Effects of proteolytic

enzymes on isolated fraction

A 0.5 ml of the fraction was digested in the presence of Trypsin (Type III-S), from bovine pancreas), Chymotrypsin (Type I-S from bovine pancreas), pepsin (from porcine stomach), carboxypeptidase A (from bovine pancreas) at concentrations of 1 mg/ml of the proteolytic enzymes. After incubating at 37°C for two hours, the enzymes were denatured by boiling for 5 minutes. The proteins were removed by centrifugation (microfuge) and the supernates adjusted to pH 7.0, evaporated to dryness and redissolved in 0.5 ml of glass distilled water and tested for its positive inotropic effect on isolated guinea pig heart. Ashin$ A partially purified fraction of 0.5 m l w a s h e a t e d in a crucible to 5000C in a kiln for 30 minutes, allowed to cool, the ash resuspended in 0.5 ml glass distilled water and centrifuged at 8740 x g for 5 minutes in Beckman microfuge. The resulting supernatant was tested for its positive inotropic effect on isolated perfused guinea pig heart. Results A crude extract was prepared from 150 gm of left ventricular tissue of pig heart after extraction of lipids, chromatographic separation and desalting. The crude extract was evaporated under reduced pressure, neutralized with Tris to pH 7.0 and dissolved to 5 ml with deionized water. The ionic content of the extract thus obtained, as measured by atomic absorption spectrophotometer and flame photometer, was 0.21, 0.27, 0.33 and 1.7 mM respectively for Mg 2+, Ca 2+, K + and Na +. The albumin extract, used as reference control, contained an ionic content of 0.88 and 2.1 mM respectively for K + and Na + and non-measurable amounts of Mg 2+ and Ca 2+. Effects of cardiac tissue extract on membrane Na +, K+-ATPase and [3H]-ouabain binding Aliquots ATPase activity tration of the Albumin extract igated.

of this DLS containing extract specifically inhibited Na +, K +in a dose dependent manner (Table i). Even at highest concenextract used, Mg2+-ATPase activity was relatively unaffected. had no effect (data not shown) on either of the ATPase invest-

Cardiac and albumin extracts were tested for their ability to compete with [3H]-ouabain for membrane binding sites as shown in Fig i. The extract seem to contain DLS that was found to compete with [3H]-ouabain binding to the membrane similar to non-labelled ouabain. Albumin extract, on the other hand, was unable to displace the bound [3H]-ouabain.

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TABLE 1 Effects of Increasing Concentrations

of DLS on ATPase Activities ATPase Activities

DLS (~i)

(% of control)

Mg 2+ ATPase

0 i0 25 50 i00

Na +, K+-ATPase

i00 102.7 99.6 93.5 89.9

i00 87.0 65.0 46.6 37.5

Control activities for Na +, K+-ATPase and MgZ+ATPase were 8.2 ± 0.4 and 19.8 ± 0.7 pmol pi/mg/hr respectively. Na +, K+-ATPase activity was almost completely inhibited by i ~M ouabain.

100

Z~ --

0

80 ,..D Z

60

z ~x3 z

40 NI 8 t~.

20

0

×

X

I

I

I

40

/,5

50

'•- - ×

I

I

I

I

I

2

4

6

8

10 12

TIME

I

i~

(MINUTES) FIG. 1

Time course displacement of specific [3H]-ouabain binding in sence of 10-6M cold ouabain ( x ~ x ) , 100pl DLS (P----e) and 500~i of extract (o--o). Specific binding (B max 12.5 pmol/mg) was carried cubating 50~g of protein with 4.2 x IO-9M [3H]-ouabain in the assay

the prealbumin out by inmedium.

Effects of pig heart extract on isolated perfused suinea pig heart Cardiac extract produced a positive inotropic response on isolated guinea pig heart in dose dependent manner (see Fig. 2). Since the DLS in the

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extract was continuously washed off by the perfusate, the response was shortlived. In another setting, the extract from a different pig heart was combined with ouabain before injection and the similarity of its DLS-like properly tested. As can be seen in Fig 3 (middle and bottom tracings), the inotropic response followed the same time course and profile as the injection of the

0~05mL 12

(g)

Froction

J

FIG. 2

Frcctj .on

0.1 rnL i2

(g)

Physiograph recordings from Langendorff preparation of isolated perfused guinea pig heart, paced at 180 beats/min and at 2 g of resting tension. The tracings show positive inotropic response to injections of cardiac extract fractions of 0.05 ml to 0.4 ml. Typical tracings from ten extractions.

i O2mL

Fraction

0.3 mL

Fraction

12

(g)

12

(g)

12

(g)

I

0

I

l iI

2 4 TIME (min)

I

l

6

8

extract alone (top tracings). However, when the concentration of ouabain was doubled (see bottom tracings), the resting tension began to increase after 5 min of continuous perfusion. That this increase in the resting tension was due to ouabain and not an effect of the crude fraction is demonstrated in Fig 4. Fig 4 shows a dose-response of ouabain on an isolated perfused guinea pig hear~ An increase in the dose of ouabain caused an increase in the inotropic response. However, when 0.2 ml of imM ouabain was injected (see bottom tracings), there was an increase in the resting tension after 5-6 min of inotropic response.

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0t mL Fraction !6

(g) 8 4 0

0-t mL

Fraction + 0.1 mL

1 mM

0uabajFI

O.lmL

FrQction+ 0 . 2 m L

lmM

Ouabain

12 (g)

lil 12

(g)

I 0

I 1

I 2

I I I 3 4 5 Time (min)

1 6

1 ?

I 8

I 9

FIG. 3 Physiograph recordings from Langendorff preparation of an isolated perfused guinea pig heart. Top tracings show positive inotropic response of a pig heart extract. The middle and the bottom tracings show time course of inotropi¢ response to combined extract and ouabain injection with increasing concentration of ouabain. The inotropic response of the crude extract was not blocked by high doses of adrenoceptor blockers such aspropran~lol(Fig 5). As can be seen, the inotropic effects of isoproterenol was completely blocked by propranolol, whereas crude fraction, containing DLS, continued to be inotropic in the propranolol treated hearts (top and bottom tracings). Effects of cardiac tissue extract on (-) 3H-DHA binding To further investigate if the cardiac tissue extract contained catecholamine residue, its effects on (-) 3H-DHA (B max 102 f mol/mg) by O.I~M propranolol is shown in Fig 6. Large aliquots of the extract were ineffective in the displacement the B-adrenoceptor ligand bound to the membrane fraction.

of

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Inotropic Substance From Heart

0-I mL

2489

.500 uM Ouaboin

12 (g)

0.1 mL I mM Ouobain 12 (g)

0 2 mL ImM Ouoboin 16 (g) 1! I 0~ I

I

0 I

I

2

I

3

I

4

I

I

I

6 7 Time (rain} 5

I

8

I

9

I

;

10 11 12

FIG. 4 Physiograph recordings from Langendorff preparation of the isolated perfused guinea pig heart, paced at 180 beats/min and at 2 g of resting tension. The tracings show a dose-response to the injections of ouabain. The zero time, as indicated by arrow, is the time when the drug was injected. Typical tracings from 6 experiments. Other Characteristics The DLS present in the heart extract is of low molecular weight (< 500 daltons) as judged by ultrafiltration membranes and gel chromatography. It is fairly stable and bears positive charge at acid pH. Ashing of the extract at 500°C destroyed the inotropic activity but treatment with protease, e.g. trypsin and other proteolytic enzymes for 2 hours did not result in a significant loss of activity. Discussion The present study documents the presence of digitalis-like substance in pig left ventricular heart tissue. The tissue extract, as prepared by the above described procedure, possessed ouabain-like activity in that it displaced [3H]ouabain from isolated membrane receptor sites, inhibited Na +, K+-ATPase and produced positive inotropic response on isolated perfused guinea pig heart. DePover et al (15) have also reported a factor in guinea pig heart extract,

2490

Inotropic Substance From Heart

01 mL Froctior

Vol. 39, No. 25, 1986

,0 05 mL luM

I~,;~r,;,:

Cg)

FIG. 5 0L I 0

I I

I 2

I 3

I I 67

I 5

I 0

I 1

I 2

I 3

I 4

Physiograph recordings from Langendorff preparation of the isolated perfused guinea pig heart. The top tracings show the inotropic responses to the injections of extracted fraction and isoproterenol. The bottom tracings show the responses of the extract and isoproterenol after B-blockade (middle tracings) with propranolol. Typical tracings from 6 experiments.

I I I 5 6 7

Time(min)

Time (rain)

0-I mL

Propronolol

8

0 ~.I 0

I I

I 2

I 3

I

4

I 5

I 6

I 7

Time (mm)

01 mL 1 uM Lsoprot.

0.1 mL

Froction

8

0 L-

l 0

l 1

l l l l l J 2 3 4 5 6 7 Time(min)

L I I I 0 1 2 3

I I 4 5

I 6

I 7

Time (min)

which displaced ~ - o u a b a i n binding and inhibited Na +, K+-ATPase. Their acetone: IN HCI, however, contained non-specific enzyme inhibitors, the reason for which is not clear. Furthermore, acetone-HCl extracts of rat (13) and guinea pig (21) brain tissue extracts have also been demonstrated to specifically inhibit Na +, K+-ATPase and compete with [3H]-ouabain binding to the membrane receptor sites. Pig heart extracts, obtained in our laboratory, produced positive inotropic response, on isolated perfused guinea pig heart, in a dose-dependent manner. In addition the inotropic responses of the extract and ouabain were found additive. The digitalis-like characteristics of the heart extract could not have been due to the residual excessive cations, as the concentration of the cations in the final extracted fraction were too low to account for any of the above digitalis-like activities. The fact that the albumin extract was unable to displace ~ u a b a i n binding and was devoid of any inotropic activity (data not shown), rules out the possible artifacts of extraction procedure.

Vol. 39, No. 25, 1986

Inotropic Substance From Heart

100~ ~ ~ ~ ~ _

2491

"~o 0

A v

BO 6O

I-

z

< 12122

"5" v

×

X

20

U_ 0

0

I

I

I

I

I

2

4

6

8

10 12

TIME

I

~

I

I

I

40

45

50

(MINUTES)

FIG. 6 Time course displacement of specific (-)3H-DHA binding in the presence of 10-7M propranolol ( x - - x ) and 200~i of DLS (o--o). Specific (-)3H-DHA binding was carried out by incubating 50~g of sarcolemmal protein with 5.0 x 10-9M (-)3H-DHA in the assay medium. It is worthwhile to point out that DLS in our extract does not exactly mimic the action of ouabain. The extracted DLS seems to have somewhat steeper doseresponse curve than does ouabain. Furthermore, increased dose of DLS did not cause any increase in the resting tension (Fig 2) as did ouabain (Fig 4). This would indicate that endogenous DLS is quite different than ouabain in some characteristics. The molecular nature of the endogenous digitalis-like factor has not been fully characterized. On the basis of positive fluorescamine reaction, Gruber et al (7) concluded that it may be a small peptide. According to LaBella (4) and Schreiber (16), the putative endogenous ligand for the digitalis receptor may include both steroids and peptides. Further studies are, however, needed to determine the true nature of the DLS in our crude fraction. Acknowledsements The author wishes to thank Greeta Kaushal for technical assistance and Mrs. M. Protosavage for manuscript preparation. This work was supported by a grant from the Medical Research Council of Canada.

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Vol.

39, No. 25, 1986

References i. 2. 3. 4. 5. 6. 7. 8. 9. 10. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

S. RINGER, J Physiol (London) 6, 361 (1885) A.J. CLARK, J Physiol (London) 47, 66 (1913) E.S. FAUST, Verhandl Schweiz Naturforch Ges Section iO, 229 (1921) F.S. LABELLA, Trends Pharmacol Sci ~, 354-355 (1982) B. CHOW, R.S. KIM, F.S. LABELLA and G. QUEEN, Brit J Pharmacol 67, 345352 (1979) R.S. KIM and F.S. LABELLA, Pharmacol Ther 14, 391-409 (1981) K.A. GRUBER, J.M. WHITAKER and W.M. BUCKALEW, Nature 287, 743-745 (1980) D. KLINGMULLER, E. WEILER and H.J. KRAMER, Klin Wochenschr 60, 1249-1253, (1982) V. SCHREIBER, F. KOLBEL, J. STEPHAN, I. GREGOROVA and T. PRIBYL, J Mol Cell C a r d i o 1 1 3 , 107-110 (1981) L. POSTON, R.B. SEWELL, S.P. WILKINSON, P.J. RICHARDSON, R. WILLIAMS, E.M. CLARKSON, G.A. MCGREGOR and H.E. WARDNER, Brit Med J 282, 847 (1981) A.D. BEYERS, L.L. SPRUYT, H.I. SEIFART, A. KRIEGLER, D.P. PARKIN and P.P. VAN JAARSVELD, S Afr Med J 65, 878-882 (1982) A. SCHWARTZ, K. WHITMER, G. GRUPP, I. GRUPP, R.J. ADAMS and SHIE-WONG LEE, Ann NY Acad Sci 402, 253-271 (1982) D. LICHTSTEIN and S. SAMUELOV, Proc Natl Acad Sci USA 79, 1453-1456 (1982) ALAIN DE POVER, European J Pharmaco199, 365-366 (1984) ALAIN DE POVER, G. CASTAWEDA-HERNANDEZ and T. GODFRAIND, Biochem Pharmacol 31, 267-271 (1982) V. SCHREIBER, J. STEPHAN, I. GREGOROVA and J. KREJCIKOVA, Biochem Pharmaco] 30, 805-806 (1981) J.C. KHATTER and R.J. HOESCHEN, Cardiovasc Res 16, 80-85 (1982) O.H. LOWRY, M.J. ROSEBROUGH, A.L. FARR and R.J. RANDALL, J Biol Chem 193, 265-275 (1951) J.C. KHATTER, J Cardiovasc Pharmacol ~, 258-261 (1985) J.S. KARLINER, P. BARNES, M. BROWN and C. DOLLERY, Eur J Pharmacol 67, 115-121 (1980) M.C. FISHMAN, Proc Natl Acad Sci USA 76, 4661-4663 (1979)