Neural factors in digitalis toxicity: effect of C-l spinal cord transection
protective
JOHN C. SOMBERG, TEUT RISLER, AND THOMAS W. SMITH Cardiovascular Division, Peter Bent Brigham Hospital, and Department Harvard Medical School, Boston, Massachusetts 02115
of Medicine,
SOMBERG,JOHN C.,TEUT RISLER, ANDTHOMAS W. SMITH. Neural factors in digitaLis toxicity: protective effect of C-l spinal cord transection. Am. J. Physiol. 235(5): H53LH536, 1978 or Am J. Physiol.: Heart Circ. Physiol. 4(5): H531-H536, 1978. -The autonomic nervous system has been shown to play a role in the genesis of cardiac rhythm disturbances. The protective effect of C-l spinal cord transection, as judged by higher cumulative ouabain dose and myocardial content of glycoside at onset of toxicity, has been cited as evidence implicating the nervous system in digitalis-induced cardiotoxicity. To define further the significance of this apparent protective effect, cats were subjected to C-l spinal cord section or were left neurally intact and [:sH]ouabain was given min) until onset of ventricular tachintravenously (1 ycardia (VT). The dose to onset of VT and the myocardial content of ouabain were significantly greater in the group with C-l cord section. In vitro active transport of the K+ analog Rb+ by myocardial samples from neurally intact cats was reduced to 54% of control at onset of VT. Significantly greater transport inhibition, to 30% of the control value, was present at VT in animals with the cord transected at the C-l level. Active transport of Rb+ at onset of VT was also significantly different between neurally intact animals and animals with C-l section when the ouabain infusion rate was doubled. Thus, C-l cord section appears to provide true protection against the emergence of overt digitalis toxicity. This effect cannot be attributed to reduced uptake of the glycoside or to reduced interaction with the putative receptor Na+-K+-ATPase.
were not a valid reflection of drug bound to cellular sites mediating toxicity, but rather included nonspecifically bound glycoside and/or an increased proportion of drug in the interstitial space. . A large body of evidence suggests that the monovalent cation transport enzyme system, sodiumand potassium-sensitive adenosine triphosphatase (NatK+-ATPase), is the locus for the direct toxic effects of digitalis on the heart (1, 7, 12, 24, 25). Accordingly, to define further the significance of the higher myocardial content of ouabain found at onset of overt toxicity after spinal cord section, activity of Na+-K+-ATPase was assessed by the active uptake of the K+ analog Rb+, determined in vitro in myocardial samples from neurally intact and denervated animals. If this enzyme system is indeed the site of direct myocardial digitalis effect, the level of monovalent cation transport inhibition should correlate with the development of arrhythmias. Further, if neurally mediated effects influence onset of arrhythmias, the level of transport inhibition at onset of arrhythmias might be altered by changes in neural input to the heart. A lesser degree of inhibition would be expected in animals with an intact nervous system and greater inhibition in animals deprived of toxicity-enhancing neural input. These hypotheses were tested in the experiments to be described.
autonomic nervous port; ouabain
METHODS
,cLg/kg
l
system;
monovalent
cation
active
trans-
SUBSTANTIAL EXPERIMENTAL WORK in reCeIIt years (11, 16, 18) the role of neural factors in modulating the arrhythmogenic effects of cardiac glycosides remains controversial. Previous studies have demonstrated that cats with spinal section at the atlantooccipital junction had a higher cumulative ouabain dose and myocardial content at the onset of toxic arrhythmias than neurally intact animals (5, 18, 29). Although spinal section produces reduction in heart rate and in both systolic and diastolic blood pressures, and therefore in myocardial blood flow, the higher myocardial content of ouabain found at toxicity in neurally deprived preparations suggests that more drug is needed to produce overt toxicity in the absence of intact neural pathways. However, the possibility remained that the higher myocardial glycoside levels at cardiotoxicity DESPITE
0363-6135/78/0000-0000$01.25
Copyright
0 1978 the American
Experimental protocols. Experiments were performed on 40 cats of either sex weighing 1.6-3.0 kg. Animals were anesthetized with sodium pentobarbital (Nembutal), 30 mg/kg, injected intraperitoneally. Tracheal cannulation was performed and animals were ventilated with room air by means of a respirator. Respiratory rate and tidal volume were adjusted to maintain arterial blood pH, PoZ, and PcoZ within normal limits for the cat (9). Body temperature was measured by means of a rectal thermometer and maintained at 3637OC with radiant heat. One femoral artery was cannulated for the continuous recording of blood pressure and for obtaining arterial blood samples for pH, PO,, and PcoZ measurements. A femoral vein was cannulated for the infusion of ouabain by a constant infusion pump. Vagotomy was performed and all other autonomic nerves leading to the heart were undisturbed as previously described (18, 29). In some animals, the spinal cord was interrupted surgically at the atlantooc-
Physiological
Society
H531
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 16, 2019.
H532
SOMBERG,
cipital junction. A period of 1 h was permitted to elapse after spinal section before drug administration was initiated. Ouabain was infused at two dose rates (1 and 2 ,ug/ kg* min) to neurally intact cats and cats with spinal transections. In another group of spinal-sectioned cats receiving ouabain 1 min, the experiment was terminated at the mean time to onset of ventricular tachycardia observed in the neurally intact group. Each of these five experimental groups consisted of six cats. Six additional cats were killed immediately after anesthesia, and myocardial active Rb+ transport was determined as described below. Rb+ transport was also determined in myocardium from two cats after all surgery had been performed and 1 h had been permitted to elapse. In an additional two animals, spinal section was performed and the Rb+ uptake determined 2 h later. To determine myocardial drug content, [3H]ouabain (New England Nuclear Corp., Boston, MA; sp act 12.5 Ci/mmol) was diluted with unlabeled ouabain in normal saline to permit delivery by constant infusion of 1 ,ug ouabain per kg body wt in a 0.2-ml volume each minute. After 1 min of sustained ventricular trachycardia, the infusion was stopped and ventricular myocardial ouabain content was determined as previously described (14). Monovalent cation transport measurements. Myocardial monovalent cation active transport was estimated by measurement of Rb+ uptake by minor modification of the method of Hougen and Smith (14). Ventricular myocardium was carefully excised and divided into approximately 1 x 2 x 5 mm samples averaging 10 mg in weight (range 3-16 mg), with epicardial to endocardial architecture preserved. The myocardial samples were placed in physiologic medium containing (in mM concentrations) KC1 4.0, NaCl 120, NaH,CO, 24, MgCl, 2.0, CaCl, 2.5, glucose 5.6, and NaH,PO, 1.1. The pH was ajusted to 7.4, with the medium gassed with 95% O,-5% C02, and the temperature maintained at 30°C. After a 5-min equilibration period, myocardial samples were transferred to individual tubes containing 1 ml of the same buffer except that ssRb+ (New England Nuclear) was added to give count rates of lo5 cpm/ml. Unlabeled RbCl was present at a final Rb+ concentration of 0.1 mM. The tubes were gassed with 95% O,-5% CO,. The myocardial tissue samples were incubated in the presence or absence of ouabain (10v3 M) for periods varying from 10 to 60 min at 30°C. After initial experiments demonstrated linear Rb+ active transport for at least 45 min under these conditions, subsequent experiments were conducted using a 30-min incubation time. At the completion of incubation, slices were rinsed and Cerenkov radiation from individual slices was determined using a Beckman model LS-133 scintillation counter. The tissue samples were gently blotted and weighed immediately after counting. Active uptake of Rb+ was determined (in nanomoles Rb+/mg wet wt per 30 min) as the difference between uptake in the presence and absence of fOa3 M ouabain. To determine if Rb+ is handled in a manner similar to pg/kg
RISLER,
AND
SMITH
I .6 -
I .4 -
l
1.2 -
,,, 0.62 k += 0.4 n IT
-
INCUBATION
I
I
I
30
45
60
TIME
(mm)
1. Time course of Rb+ transport in left ventricular myocardial samples. Left ventricular myocardial slices were incubated at 30°C for 5, 15; 30, 45, or 60 min (20 samples in each group) in physiologic buffer containing 4.0 mM K+ and 0.1 mM Rb’, with ssRb+ present as tracer (lo5 cpm/ml). Upper curve (O-----O> shows total Rb+ uptake in absence of ouabain and the lower curve (A-A) uptake in presence of lo-:$ M ouabain. The difference is plotted o) as active Rb+ transport (nmol/mg wet wt). coFIG.
K+ under the conditions of these experiments, tracer quantities of 42K+ were also added to the incubation medium in separate experiments. After incubation, as described above, myocardial samples were individually counted and then recounted 7 days later when the 42K+ activity (tt 12.4 h) had decayed to less than 0.006% of the activity present at the time of initial counting. In this way, total and ouabain-inhibitable uptakes of K+ and Rb+ could be determined independently in each sample. The significance of differences between mean values was determined by Student’s t test. Experimental results are reported as means t SE. RESULTS
Time course of Rb+ uptake. Experiments were performed using variable periods of incubation to determine the time course of Rb+ transport. Linearity of active transport was observed for at least 45 min, as shown in Fig. 1. The active uptake of Rb+ was subsequently determined in all experiments using an incubation period of 30 min. Additional experiments were performed to validate the use of Rb+ as a marker for K+ transport. If active
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 16, 2019.
NEURAL
EFFECTS
OF DIGITALIS
H533
TOXICITY
transport of both ionic species is identical, the predicted ratio of K+ uptake to Rb+ uptake should be 4 to 0.1 or 40 to 1. Two experiments summarized in Table 1 yielded ratios of 38:l and 39:l for K+-to-Rb+ active uptake. The Rb+ transport rate was not affected by duration of anesthesia and surgery alone. Control active Rb+ uptake was 0.63 t 0.04 nmol/mg=30 min, and was essentially unchanged at 0.63 t 0.01 nmol/mg*30 min after spinal section (without ouabain). Thus, active myocardial uptake of Rb+ was unchanged from the control state by duration of anesthesia or spinal cord section. Hemodynamic effects of spinal cord section. The effects of spinal section on heart rate and blood pressure are summarized in Table 2. As expected, spinal section caused a reduction in both heart rate and arterial blood pressure (5, 18, 29). The mean base-line systolic blood pressure of spinal-sectioned cats receiving 2 ,ug/kg min of ouabain was somewhat higher then that of spinalthe sectioned cats receiving 1 pg/kg min, although diastolic pressures were similar. Animals with spinal section receiving the more rapid infusion rate had a significantly greater degree of heart rate reduction in response to ouabain than the group receiving 1 pg/ kgemin (P < 0.05).
Effects of spinal cord section on ouabain tolerance and myocardial R b+ transport. Spinal section at the atlantooccipital junction increased both the dose of ouabain needed to produce ventricular tachycardia (VT) and the myocardial content of ouabain at onset of VT compared with neurally intact preparations (Table 3). C-l spinal cord section increased the dose of ouabain at a 1 pg/kg min infusion rate from 78 t 3 (SE) to 117 t 9 pglkg (P < 0.01). Myocardial ouabain content at onset of VT was increased from 0.67 t 0.06 in controls to 1.25 -+ 0.09 pmol/mg wet wt (P < 0.001) in cats with C-l cord section. Spinal section also significantly decreased the level of active uptake of Rb+ at onset of VT by 44% from 0.34 t 0.04 nmol/mg 30 min in the neurally intact preparation to 0.19 t 0.01 nmol/mg*30 min (P < 0.01). Rubidium active transport was 0.63 t 0.04 nmol/mge 30 ‘min in animals not given ouabain. Thus, active transport of Rb+ was decreased to 54% of control in neurally intact cats at the onset of ouabain-induced VT. Significantly greater transport inhibition, to 30% of the control value, was present at onset of VT in animals with C-l spinal cord transection, indicating that the higher myocardial ouabain content does correlate with greater inhibition of monovalent cation active transport. When experiments with spinal-sectioned cats receiving ouabain at a rate of 1 pg/kg min were terminated at a total dose of 78 pg/kg, the myocardial content of ouabain and the degree of inhibition of active Rb+ transport were similar to data from the neurally intact group (Table 3). The active uptake of Rb+ was 0.34 t 0.04 nmol/mg 30 min in the neurally intact group and 0.30 t 0.01 nmol/mg.30 min in the spinal-sectioned group killed after 78 min. Thus, the neurally intact preparations had sustained ventricular tachycardia at a time when the spinal preparations had a normal sinus rhythm, with essentially the same myocardial l
l
l
l
1. Uptake ofK+ ventricular myocardial
TABLE
Total nmol/mg
Rb+ K+
and Rb+ by left strips
Uptake, wet wt * 30 min
Active nmol/mg
0.76 t 0.06 28.0 IL 5.3
Uptake, wet wt - 30 min
l
Passive nmol/mg
0.62 + 0.04 23.7 k 2.7
Uptake, wet wt * 30 min
l
0.14 2 0.02 4.26 ix 0.61
Values are means + SE. Forty myocardial strips were employed, weighing 3-16 mg; 20 strips were analyzed in the presence of 42K+ and 86Rb+, and 20 in the presence of 42K+, 86Rb+, and ouabain (lOA Ml.
TABLE 2. Heart
rate and arterial
Postva otomy and Cor cf Section, when Performed
Initial Determination HR
blood pressure
BP
HR
BP
15 min HR
30 min BP
HR
45 min BP
HR
75 min BP
Ouabain 1 p.g/kg min 196 209 169 193 217 +16 +6 +15 131 121 k6 +3
105 min
HR
BP
173 +3 119 +6
211 220
178 217 116 +9
87 +9 55 +8
147 +12
77 +17 54 +6
120 min
HR
BP
HR
BP
160 +14
84 +ll 51 +lO
161 +15
113 a2 55 +5
l
Neurally
204 +13
170 +15 125 +9
208 *13
183 +8 131 +5
204 +13
195 +6
178 It12 126 +8
162 +lO
69 +15 52 +5
152 +15
207 +12
143 +_7 112 +4
217 &lo
163 &lo 111 &lo
195 +8
217 k12
179 +ll 122 +9
155 k6
107 +7 56 27
140 t8
intact C-l cord sectioned
Neurally intact
C-l cord sectioned
Values are means + SE for 6 cats in each group. diastolic blood pressure are given in BP columns.
87 28 54 +7
151 tll
90 +8 54 +8
154 +ll
Ouabain 2 p&kg- min 163 195 160 182 +6 +7 +3 a4 110 104 *lo +9 104 t2 50 +6
In 6 columns
129 +5
at right,
100 +3 53 +6
128 +ll
headings
162 +ll 97 +14 102 +8 53 +4
are time after beginning
ouabain
infusion.
Systolic
and
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 16, 2019.
H534
SOMBERG,
3. Influence of spinal cord transection: infusion rate 1 N/kg* min
TABLE
ouabain
Dose
Active Rb+ Uptake, nmol/ mg * 30 min
Myocardial Ouabain Content, pmol/mg wet wt
78 + 3
0.34 AI 0.04
0.67 rt 0. 06
117 z!z 9*
0.19 I!z 0.01*
1.25 t 0.09*
O-30 t 0.01t
0.56 + 0.09t
of Ouabain VT i%kz
Ouabain, 1 pg/ kg min Ouabain, 1 pg/ kg-min, + C-l cord section Ouabain, 1 pgl kg min (stopped at 78 pg/kg) + C-l cord section
to
l
l
Values given are means SE for groups of 6 animals. * Sign% cantly different from neurally intact group receiving ou .abain (P < 0.01 1. t Significantly diffe rent from C-l cord section (P < 0.01) but not significantly different from neurally intact group. TABLE
ouabain
4. hfluence of spinal cord transection: infusion rate 2 M/kg- min Neurally
Dose of ouabain to VT, /%/kg Active Rb+ uptake at VT onset, nmol/mg wet wt 30 min Myocardial ouabain content at VT onset, pmol/mg wet wt
Intact
Significance
C-l
Cord
Section
94 Ik 5
NS
98 It 3
0.32 t 0.05
P < 0.025
0.19 z!I 0.01
0.59 ?I 0.05
P < 0.001
1.07 Ik 0.09
l
drug content and inhibition of monovalent cation active transport. In an additional set of experiments, ouabain was infused at 2 pg/kg min, twice the infusion rate studied above. At this rate of infusion, spinal section did not alter the cumulative dose required to produce ventricular tachycardia. However, in the neurally deprived (C-l sectioned) group, a higher myocardial ouabain content was present at onset of VT, and this was accompanied by significantly greater monovalent cation transport inhibition, to 30% of control at onset of VT, compared with neurally intact animals (Table 4). The arrhythmogenic dose of ouabain was 94 t 5 pg/kg in the neurally intact group and 98 t 3 rug/kg in the group with C-l cord section. However, active Rb+ transport was reduced from 0.32 t 0.05 nmol/mg 30 min in the neurally intact series to 0.19 t 0.01 nmol/ mg 30 min in the C-l cord section series, a 41% reduction, similar to the change of 44% observed in experiments employing a 1 pg/kg min ouabain infusion rate. l
l
l
l
DISCUSSION
Substantial evidence suggests that the sympathetic nervous system plays a part in the genesis of disorders of cardiac rhythm produced by digitalis glycosides (see reviews by Levitt et al. (17, 19) and Roberts et al. (26)]. Cardiac glycoside-induced augmented activity in sympathetic nerves leading to the heart has been reported by a number of workers (10, 11, 21). The surgical
RISLER,
AND
SMITH
interruption of this augmented activity by spinal cord transection at the atlantooccipital junction has been shown to provide protection against digitalis-induced arrhythmias in our study as well as in previous work (4, 8, 11, 18, 29). However, the protective effect of spinal .cord section has been difficult to interpret because of concomitant reduction of heart rate and arterial blood pressure. These effects have been shown to influence myocardial uptake of cardiac glycosides (2, 13, 20, 21). Previous studies have, therefore, utilized tritium-labeled digitalis preparations to determine if the rate of myocardial uptake was reduced in the spinal-sectioned animals and if a higher level of digitalis content in the myocardium was in fact required to produce arrhythmias when the myocardial sympathetic neural innervation had been interrupted (4, 18, 29). This study confirms that a higher myocardial content of digitalis is found in the neurally ablated group at the onset of ventricular arrhythmias. We further addressed the significance of the higher myocardial content in the neurally deprived preparation by determinating the degree of inhibition of monovalent cation transport associated with higher myocardial levels of ouabain. The potassium analog Rb+ is similar to K+ in its transport properties (3) and has been used as an indicator of Na+-K+-ATPase-dependent sodium and potassium transport in red blood cells (27, ZS), rat brain (3), guinea pig left atrium (6, 27), guinea pig ventricular slices (X5), and canine left ventricular samples (14). This study used the measurement of Rb+ uptake as an index of Na’-K+-ATPase-dependent monovalent cation transport in samples of cat ventricular myocardium. The results with left ventricular myocardial slices demonstrate linearity of Rb+ uptake beyond the time period studied (30 min). Furthermore, under the conditions of these experiments, active Rb+ uptake was quite similar to active K+ uptake (Table 1). Our results in this cat experimental model demonstrate a greater inhibition of monovalent cation active transport in left ventricular myocardium at the onset of overt ouabain cardiotoxicity in animals in which the sympathetic neural input to the heart is interrupted by C-l spinal cord section. These data indicate that the higher myocardial drug content found in the spinal preparation at onset of VT is biologically significant in that the drug has interacted specifically with Na+-K+ATPase, the putative receptor site for toxic effects of digitalis glycosides (1, 7, 12, 24, 25). The greater inhibition of Rb+ transport at onset of toxicity found in animals with spinal cord transection provides indirect support for the hypothesis that inhibition of Na+-K+-ATPase is related to the direct toxic effects of digitalis on the heart, although the present, data do not establish a causal relationship. With C-l cord section removing sympathetic neural influences on the heart, cardiac glycoside-induced ventricular arrhythmias emerge at a higher myocardial content and at greater levels of inhibition of active monovalent cation transport. The finding of greater transport inhibition by ouabain at the onset of sustained cardiotoxicity in cord-sectioned animals is consistent with the thesis that the sympathetic autonomic nervous system
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 16, 2019.
NEURAL
EFFECTS
OF DIGITALIS
H535
TOXICITY
facilitates the development of overt toxicity. This concept of arrhythmia facilitation is further supported by the observation that at similar levels of monovalent cation transport inhibition, neurally deprived animals are in sinus rhythm while neurally intact animals are in sustained ventricular tachycardia (Table 3). The similar cumulative ouabain doses to onset of VT with and without spinal section at the higher infusion rate of ouabain is in agreement with the previous report of Cagin et al. (5). The data presented here, however, show that the myocardial content of glycoside was higher and the inhibition of monovalent cation transport greater in the neurally deprived group, demonstrating a dependence of myocardial glycoside levels and monovalent cation active transport inhibition at onset of overt cardiotoxicity on the state of the nervous system. Thus the failure of cumulative doses per se at higher infusion rates to reflect changes in the mechanisms causing or facilitating cardiotoxicity could explain many of the past inconsistencies in the literature. The failure to find protection by antiadrenergic interin ventions in the past (23, 30, 31) may be explained part by this phenomenon. The determination of inhibition of myocardial monovalent cation active transport provides information over and above that obtained from myocardial cardiac glycoside levels, as exemplified by the data in Tables 3 and 4. The degree of Rb+ active uptake is essentially the same at onset of VT at the two rates of infusion in both spinal-sectioned and neurally intact animals,
whereas total dose and myocardial ouabain content show considerable variation. Thus the failure of cumulative dose during constant infusion to correlate with the onset of cardiotoxicity and its failure to reflect modifying influences of the nervous system can be reconciled in these experimental studies by the determination of active monovalent cation transport. These data provide further support for the concept that the intactness of the nervous system influences the response of the heart to a given dose of cardiac glycoside. Cardiotoxicity precipitated by ouabain involved an interaction of neurally mediated and direct cardiac actions of the glycoside in this experimental model. With the nervous system intact, bursts of nonuniform neural input appear to augment the direct cardiotoxic effects of digitalis (16), setting the stage for the development of potentially lethal cardiac arrhythmias. With the sympathetic neural input interrupted in spinalsectioned animals, higher myocardial ouabain content and greater inhibition of monovalent cation transport are observed at onset of ventricular arrhythmias, indicating that more receptor-bound drug is needed to produce overt cardiotoxicity. This investigation was supported by National Heart, Lung, and Blood Institute Fellowship lF32HL05542-01 and Grant HL-18003, by American Heart Association Award 76-597, and by the Paul-Martini Foundation. Received
24 April
1978; accepted
in final form 5 July 1978.
REFERENCES 1. AKERA, T., F. S. LARSEN, AND T. M. BRODY. The effect of ouabain on sodium and potassium activated adenosine triphosphate from the heart of several mammalian species. J. PharmacoZ. Exptl. Therap. 170: 17-26, 1969. 2. BELLER, G. A., T. W. SMITH, AND W. B. HOOD, JR. Altered distribution of tritiated digoxin in the infarcted canine left ventricle. CircuZation 46: 572-579, 1972. 3. BERNSTEIN, J. C., AND Y. ISRAEL. Active transport of Rbs” in human red cells and rat brain slices. J. Pharmacol. Exptl. Therap. 174: 323-329, 19’70. 4. CAGIN, N., J. C. SOMBERG, H. BOUNOUS, T. MITTAG, A. RAINES, AND B. LEVITT. The influence of spinal cord transection on the capacity of digitoxin to induce cardiotoxicity. Arch. Intern. Pharmacodyn. Therap. 207: 370-374, 1974. 5. CAGIN, N., J. C. SOMBERG, H. BOUNOUS, T. MITTAG, A. RAINES, AND B. LEVITT. Ouabain cardiotoxicity, a reassessment of methodology. Arch. Intern. Pharmacodyn. Therap. 224: 230-238,1976. 6. CURFMAN, G. D., T. J. CROWLEY, AND T. W. SMITH. Thyroidinduced alterations in myocardial sodium and potassium activated adenosine triphosphatase, monovalent cation active transport, and cardiac glycoside binding. J. CZin. Invest. 59: 586-590, 1977. 7. DUTTA, S., H. M. RHEE, AND B. H. MARKS. H”-ouabain accumulation and Na+, K+-ATPase activities in relation to therapeutic, toxic and lethal doses of ouabain in dogs. In: Recent Advances in Studies on Cardiac Structure and MetaboZism (Myocardial BioZogy), edited by N. S. Dhalla. Baltimore, MD: University Park Press, 1974, vol. 4, p. 119-130. 8. ERLIJ, D., AND R. MENDEZ. The modification of digitalis intoxication by excluding adrenergic influences on the heart. J. PharmacoZ. ExptZ. Therap. 144: 97-103, 19t,d. 9. FINK, B., AND A. SCHOOLMAN. Arterial blood acid base balance in unrestrained walking cats. Proc. Sot. Exptl. BioZ. Med. 112: 328-330, 1963. 10. GILLIS, R. A. Cardiac sympathetic nerve activity: changes induced bv ouabain and nronranolol. Science 166: 508-510. 1969.
11. GILLIS, R. ‘A.) A. RAINES, Y. J. SOHN, B. LEVITT, AND G. STANDAERT. Neuroexcitatory effects of digitalis and their role in the development of cardiac arrhythmias. J. PharmacoZ. Exptl. Therap. 183: 154-168, 1972. 12. GOLDSTEIN, R. E., S. C. PENZOTTI, K. S. KUEHL, K. H. PRINDLE, C. A. HALL, E. D. TITUS, AND S. E. EPSTEIN. Correlation of antiarrhythmic effects of diphenylhydantoin with digoxin induced changes in myocardial contractility, sodium potassium adenosine triphosphatase activity, and potassium efflux. CircuZation Res. 33: 175-182, 1973. 13. HOPKINS, B. E., R. R. TAYLOR, C. HENDERSON, AND P. BURROWS. Digoxin distribution in the dog’s left ventricle in the presence of coronary artery ligation. J. MOL. CeZZuZar CardioZ. 5: 197-203, 1973. 14. HOUGEN, T. J., AND T. W. SMITH. Inhibition of myocardial monovalent cation active transport by subtoxic doses of ouabain in the dog. Circulation Res. 42: 856-863, 1978. 15. Ku, D. D., T. AKERA, C. L. PEW, AND T. M. BRODY. Cardiac glycosides: correlations among Na+,K+-ATPase, sodium pump and contractility in the guinea pig heart. Arch. Exptl. PathoZ. PharmacoZ. 285: 185-200, 1974. 16. LATHERS, C. M., J. ROBERTS, AND G. J. KELLIHER. Correlation of ouabain-induced arrhythmia and nonuniformity in the histamine-induced discharge of cardiac sympathetic nerves. J. PharmacoZ. Exptl. Therap. 203: 467-479, 1977. 17. LEVITT, B., N. CAGIN, J. KLEID, J. SOMBERG, R. GILLIS. The role of the nervous system in the genesis of cardiac rhythm disorders. Am. J. CardioZ. 37: 1111-1113, 1976. 18. LEVITT, B., N. A. CAGIN, J. SOMBERG, H. BOUNOUS, T. MITTAG, AND A. RAINES. Alteration of the effects of distribution of ouabain by spinal cord transection in the cat. J. PharmacoZ. Exptl. Therap. 185: 24-28, 1973. 19. LEVITT, B., N. A. CAGIN, J. C. SOMBERG, AND J. KLEID. Neural basis of the genesis and control of digitalis arrhythmias. Cardiology 61: 50-60, 1976. - 20. LLOYD, B. L., AND R. R. TAYLOR. Augmentation of myocardial
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 16, 2019.
H536
21. 22.
23.
24. 25.
26.
digoxin concentration in hemorrhagic shock. CircuZation 51: 718-722, 1975. LLOYD, B. L., AND R. R. TAYLOR. Influence of myocardial mechanical activity and coronary blood flow on myocardial digoxin uptake. Cardiovascular Res. 10: 487-493, 1976. MCLAIN, P. Effects of cardiac glycoside on spontaneous efferent activity in vagus and sympathetic nerves of cats. Intern. J. Neuropharmacol. 8: 379-387, 1969. MORROW, D. H., T. E. GAFFNEY, AND E. BRAUNWALD. Studies on digitalis. VIII. Effect of autonomic innervation and of myocardial catecholamine stores upon the cardiac action of ouabain. J. PharmacoZ. ExptZ. Therap. 140: 236-242, 1963. OKITA, G. T. Dissociation of Na+,K+-ATPase inhibition from digitalis intropy. Federation Proc. 36: 22252230, 1977. RHEE, H. M., S. DUTTA, AND B. H. MARKS. Cardiac Na+,K+ATPase activity during positive inotropic and toxic actions of ouabain. European J. PharmacoZ. 37: 141-153,1976. ROBERTS, J., G. J. KELLIHER, AND C. M. LATHERS. Role of
SOMBERG,
27. 28.
29.
30. 31.
RISLER,
AND
SMITH
adrenergic influences in digitalis-induced ventricular arrhythmia. Life Sci. 18: 665-678, 1976. SMITH, T. W., AND H. WAGNER, JR. Effects of (Na+ + K+)ATPase specific antibodies on enzymatic activity and monovalent cation transport. J. Membrane BioZ. 25: 341-360, 1975. SMITH, T. W., H. WAGNER, JR., J. E. MARKIS, AND M. YOUNG. Studies on the localization of the cardiac glycoside receptor. J. CZin. Invest. 51: 1777-1789, 1972. SOMBERG, J. C., H. BOUNOUS, N. CAGIN, C. ANAGNOSTROPOULOS, AND B. LEVITT. The influence of prostaglandins E, and E, on ouabain cardiotoxicity in the cat. J. PharmacoZ. ExptZ. Therap. 203: 480-484, 1977. Digitalis WILLIAM, V. L., T. COOPER, AND C. R. HANLON. tolerance of the denervated heart (Abstract). Federation Proc. 24: 486, 1965. YELONSKY, J., AND R. ERVIN. The effect of ouabain on cardiac automaticity in reserpine-pretreated dogs. Am. Heart J. 62: 687689, 1961.
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 16, 2019.
protective
JOHN C. SOMBERG, TEUT RISLER, AND THOMAS W. SMITH Cardiovascular Division, Peter Bent Brigham Hospital, and Department Harvard Medical School, Boston, Massachusetts 02115
of Medicine,
SOMBERG,JOHN C.,TEUT RISLER, ANDTHOMAS W. SMITH. Neural factors in digitaLis toxicity: protective effect of C-l spinal cord transection. Am. J. Physiol. 235(5): H53LH536, 1978 or Am J. Physiol.: Heart Circ. Physiol. 4(5): H531-H536, 1978. -The autonomic nervous system has been shown to play a role in the genesis of cardiac rhythm disturbances. The protective effect of C-l spinal cord transection, as judged by higher cumulative ouabain dose and myocardial content of glycoside at onset of toxicity, has been cited as evidence implicating the nervous system in digitalis-induced cardiotoxicity. To define further the significance of this apparent protective effect, cats were subjected to C-l spinal cord section or were left neurally intact and [:sH]ouabain was given min) until onset of ventricular tachintravenously (1 ycardia (VT). The dose to onset of VT and the myocardial content of ouabain were significantly greater in the group with C-l cord section. In vitro active transport of the K+ analog Rb+ by myocardial samples from neurally intact cats was reduced to 54% of control at onset of VT. Significantly greater transport inhibition, to 30% of the control value, was present at VT in animals with the cord transected at the C-l level. Active transport of Rb+ at onset of VT was also significantly different between neurally intact animals and animals with C-l section when the ouabain infusion rate was doubled. Thus, C-l cord section appears to provide true protection against the emergence of overt digitalis toxicity. This effect cannot be attributed to reduced uptake of the glycoside or to reduced interaction with the putative receptor Na+-K+-ATPase.
were not a valid reflection of drug bound to cellular sites mediating toxicity, but rather included nonspecifically bound glycoside and/or an increased proportion of drug in the interstitial space. . A large body of evidence suggests that the monovalent cation transport enzyme system, sodiumand potassium-sensitive adenosine triphosphatase (NatK+-ATPase), is the locus for the direct toxic effects of digitalis on the heart (1, 7, 12, 24, 25). Accordingly, to define further the significance of the higher myocardial content of ouabain found at onset of overt toxicity after spinal cord section, activity of Na+-K+-ATPase was assessed by the active uptake of the K+ analog Rb+, determined in vitro in myocardial samples from neurally intact and denervated animals. If this enzyme system is indeed the site of direct myocardial digitalis effect, the level of monovalent cation transport inhibition should correlate with the development of arrhythmias. Further, if neurally mediated effects influence onset of arrhythmias, the level of transport inhibition at onset of arrhythmias might be altered by changes in neural input to the heart. A lesser degree of inhibition would be expected in animals with an intact nervous system and greater inhibition in animals deprived of toxicity-enhancing neural input. These hypotheses were tested in the experiments to be described.
autonomic nervous port; ouabain
METHODS
,cLg/kg
l
system;
monovalent
cation
active
trans-
SUBSTANTIAL EXPERIMENTAL WORK in reCeIIt years (11, 16, 18) the role of neural factors in modulating the arrhythmogenic effects of cardiac glycosides remains controversial. Previous studies have demonstrated that cats with spinal section at the atlantooccipital junction had a higher cumulative ouabain dose and myocardial content at the onset of toxic arrhythmias than neurally intact animals (5, 18, 29). Although spinal section produces reduction in heart rate and in both systolic and diastolic blood pressures, and therefore in myocardial blood flow, the higher myocardial content of ouabain found at toxicity in neurally deprived preparations suggests that more drug is needed to produce overt toxicity in the absence of intact neural pathways. However, the possibility remained that the higher myocardial glycoside levels at cardiotoxicity DESPITE
0363-6135/78/0000-0000$01.25
Copyright
0 1978 the American
Experimental protocols. Experiments were performed on 40 cats of either sex weighing 1.6-3.0 kg. Animals were anesthetized with sodium pentobarbital (Nembutal), 30 mg/kg, injected intraperitoneally. Tracheal cannulation was performed and animals were ventilated with room air by means of a respirator. Respiratory rate and tidal volume were adjusted to maintain arterial blood pH, PoZ, and PcoZ within normal limits for the cat (9). Body temperature was measured by means of a rectal thermometer and maintained at 3637OC with radiant heat. One femoral artery was cannulated for the continuous recording of blood pressure and for obtaining arterial blood samples for pH, PO,, and PcoZ measurements. A femoral vein was cannulated for the infusion of ouabain by a constant infusion pump. Vagotomy was performed and all other autonomic nerves leading to the heart were undisturbed as previously described (18, 29). In some animals, the spinal cord was interrupted surgically at the atlantooc-
Physiological
Society
H531
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 16, 2019.
H532
SOMBERG,
cipital junction. A period of 1 h was permitted to elapse after spinal section before drug administration was initiated. Ouabain was infused at two dose rates (1 and 2 ,ug/ kg* min) to neurally intact cats and cats with spinal transections. In another group of spinal-sectioned cats receiving ouabain 1 min, the experiment was terminated at the mean time to onset of ventricular tachycardia observed in the neurally intact group. Each of these five experimental groups consisted of six cats. Six additional cats were killed immediately after anesthesia, and myocardial active Rb+ transport was determined as described below. Rb+ transport was also determined in myocardium from two cats after all surgery had been performed and 1 h had been permitted to elapse. In an additional two animals, spinal section was performed and the Rb+ uptake determined 2 h later. To determine myocardial drug content, [3H]ouabain (New England Nuclear Corp., Boston, MA; sp act 12.5 Ci/mmol) was diluted with unlabeled ouabain in normal saline to permit delivery by constant infusion of 1 ,ug ouabain per kg body wt in a 0.2-ml volume each minute. After 1 min of sustained ventricular trachycardia, the infusion was stopped and ventricular myocardial ouabain content was determined as previously described (14). Monovalent cation transport measurements. Myocardial monovalent cation active transport was estimated by measurement of Rb+ uptake by minor modification of the method of Hougen and Smith (14). Ventricular myocardium was carefully excised and divided into approximately 1 x 2 x 5 mm samples averaging 10 mg in weight (range 3-16 mg), with epicardial to endocardial architecture preserved. The myocardial samples were placed in physiologic medium containing (in mM concentrations) KC1 4.0, NaCl 120, NaH,CO, 24, MgCl, 2.0, CaCl, 2.5, glucose 5.6, and NaH,PO, 1.1. The pH was ajusted to 7.4, with the medium gassed with 95% O,-5% C02, and the temperature maintained at 30°C. After a 5-min equilibration period, myocardial samples were transferred to individual tubes containing 1 ml of the same buffer except that ssRb+ (New England Nuclear) was added to give count rates of lo5 cpm/ml. Unlabeled RbCl was present at a final Rb+ concentration of 0.1 mM. The tubes were gassed with 95% O,-5% CO,. The myocardial tissue samples were incubated in the presence or absence of ouabain (10v3 M) for periods varying from 10 to 60 min at 30°C. After initial experiments demonstrated linear Rb+ active transport for at least 45 min under these conditions, subsequent experiments were conducted using a 30-min incubation time. At the completion of incubation, slices were rinsed and Cerenkov radiation from individual slices was determined using a Beckman model LS-133 scintillation counter. The tissue samples were gently blotted and weighed immediately after counting. Active uptake of Rb+ was determined (in nanomoles Rb+/mg wet wt per 30 min) as the difference between uptake in the presence and absence of fOa3 M ouabain. To determine if Rb+ is handled in a manner similar to pg/kg
RISLER,
AND
SMITH
I .6 -
I .4 -
l
1.2 -
,,, 0.62 k += 0.4 n IT
-
INCUBATION
I
I
I
30
45
60
TIME
(mm)
1. Time course of Rb+ transport in left ventricular myocardial samples. Left ventricular myocardial slices were incubated at 30°C for 5, 15; 30, 45, or 60 min (20 samples in each group) in physiologic buffer containing 4.0 mM K+ and 0.1 mM Rb’, with ssRb+ present as tracer (lo5 cpm/ml). Upper curve (O-----O> shows total Rb+ uptake in absence of ouabain and the lower curve (A-A) uptake in presence of lo-:$ M ouabain. The difference is plotted o) as active Rb+ transport (nmol/mg wet wt). coFIG.
K+ under the conditions of these experiments, tracer quantities of 42K+ were also added to the incubation medium in separate experiments. After incubation, as described above, myocardial samples were individually counted and then recounted 7 days later when the 42K+ activity (tt 12.4 h) had decayed to less than 0.006% of the activity present at the time of initial counting. In this way, total and ouabain-inhibitable uptakes of K+ and Rb+ could be determined independently in each sample. The significance of differences between mean values was determined by Student’s t test. Experimental results are reported as means t SE. RESULTS
Time course of Rb+ uptake. Experiments were performed using variable periods of incubation to determine the time course of Rb+ transport. Linearity of active transport was observed for at least 45 min, as shown in Fig. 1. The active uptake of Rb+ was subsequently determined in all experiments using an incubation period of 30 min. Additional experiments were performed to validate the use of Rb+ as a marker for K+ transport. If active
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 16, 2019.
NEURAL
EFFECTS
OF DIGITALIS
H533
TOXICITY
transport of both ionic species is identical, the predicted ratio of K+ uptake to Rb+ uptake should be 4 to 0.1 or 40 to 1. Two experiments summarized in Table 1 yielded ratios of 38:l and 39:l for K+-to-Rb+ active uptake. The Rb+ transport rate was not affected by duration of anesthesia and surgery alone. Control active Rb+ uptake was 0.63 t 0.04 nmol/mg=30 min, and was essentially unchanged at 0.63 t 0.01 nmol/mg*30 min after spinal section (without ouabain). Thus, active myocardial uptake of Rb+ was unchanged from the control state by duration of anesthesia or spinal cord section. Hemodynamic effects of spinal cord section. The effects of spinal section on heart rate and blood pressure are summarized in Table 2. As expected, spinal section caused a reduction in both heart rate and arterial blood pressure (5, 18, 29). The mean base-line systolic blood pressure of spinal-sectioned cats receiving 2 ,ug/kg min of ouabain was somewhat higher then that of spinalthe sectioned cats receiving 1 pg/kg min, although diastolic pressures were similar. Animals with spinal section receiving the more rapid infusion rate had a significantly greater degree of heart rate reduction in response to ouabain than the group receiving 1 pg/ kgemin (P < 0.05).
Effects of spinal cord section on ouabain tolerance and myocardial R b+ transport. Spinal section at the atlantooccipital junction increased both the dose of ouabain needed to produce ventricular tachycardia (VT) and the myocardial content of ouabain at onset of VT compared with neurally intact preparations (Table 3). C-l spinal cord section increased the dose of ouabain at a 1 pg/kg min infusion rate from 78 t 3 (SE) to 117 t 9 pglkg (P < 0.01). Myocardial ouabain content at onset of VT was increased from 0.67 t 0.06 in controls to 1.25 -+ 0.09 pmol/mg wet wt (P < 0.001) in cats with C-l cord section. Spinal section also significantly decreased the level of active uptake of Rb+ at onset of VT by 44% from 0.34 t 0.04 nmol/mg 30 min in the neurally intact preparation to 0.19 t 0.01 nmol/mg*30 min (P < 0.01). Rubidium active transport was 0.63 t 0.04 nmol/mge 30 ‘min in animals not given ouabain. Thus, active transport of Rb+ was decreased to 54% of control in neurally intact cats at the onset of ouabain-induced VT. Significantly greater transport inhibition, to 30% of the control value, was present at onset of VT in animals with C-l spinal cord transection, indicating that the higher myocardial ouabain content does correlate with greater inhibition of monovalent cation active transport. When experiments with spinal-sectioned cats receiving ouabain at a rate of 1 pg/kg min were terminated at a total dose of 78 pg/kg, the myocardial content of ouabain and the degree of inhibition of active Rb+ transport were similar to data from the neurally intact group (Table 3). The active uptake of Rb+ was 0.34 t 0.04 nmol/mg 30 min in the neurally intact group and 0.30 t 0.01 nmol/mg.30 min in the spinal-sectioned group killed after 78 min. Thus, the neurally intact preparations had sustained ventricular tachycardia at a time when the spinal preparations had a normal sinus rhythm, with essentially the same myocardial l
l
l
l
1. Uptake ofK+ ventricular myocardial
TABLE
Total nmol/mg
Rb+ K+
and Rb+ by left strips
Uptake, wet wt * 30 min
Active nmol/mg
0.76 t 0.06 28.0 IL 5.3
Uptake, wet wt - 30 min
l
Passive nmol/mg
0.62 + 0.04 23.7 k 2.7
Uptake, wet wt * 30 min
l
0.14 2 0.02 4.26 ix 0.61
Values are means + SE. Forty myocardial strips were employed, weighing 3-16 mg; 20 strips were analyzed in the presence of 42K+ and 86Rb+, and 20 in the presence of 42K+, 86Rb+, and ouabain (lOA Ml.
TABLE 2. Heart
rate and arterial
Postva otomy and Cor cf Section, when Performed
Initial Determination HR
blood pressure
BP
HR
BP
15 min HR
30 min BP
HR
45 min BP
HR
75 min BP
Ouabain 1 p.g/kg min 196 209 169 193 217 +16 +6 +15 131 121 k6 +3
105 min
HR
BP
173 +3 119 +6
211 220
178 217 116 +9
87 +9 55 +8
147 +12
77 +17 54 +6
120 min
HR
BP
HR
BP
160 +14
84 +ll 51 +lO
161 +15
113 a2 55 +5
l
Neurally
204 +13
170 +15 125 +9
208 *13
183 +8 131 +5
204 +13
195 +6
178 It12 126 +8
162 +lO
69 +15 52 +5
152 +15
207 +12
143 +_7 112 +4
217 &lo
163 &lo 111 &lo
195 +8
217 k12
179 +ll 122 +9
155 k6
107 +7 56 27
140 t8
intact C-l cord sectioned
Neurally intact
C-l cord sectioned
Values are means + SE for 6 cats in each group. diastolic blood pressure are given in BP columns.
87 28 54 +7
151 tll
90 +8 54 +8
154 +ll
Ouabain 2 p&kg- min 163 195 160 182 +6 +7 +3 a4 110 104 *lo +9 104 t2 50 +6
In 6 columns
129 +5
at right,
100 +3 53 +6
128 +ll
headings
162 +ll 97 +14 102 +8 53 +4
are time after beginning
ouabain
infusion.
Systolic
and
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 16, 2019.
H534
SOMBERG,
3. Influence of spinal cord transection: infusion rate 1 N/kg* min
TABLE
ouabain
Dose
Active Rb+ Uptake, nmol/ mg * 30 min
Myocardial Ouabain Content, pmol/mg wet wt
78 + 3
0.34 AI 0.04
0.67 rt 0. 06
117 z!z 9*
0.19 I!z 0.01*
1.25 t 0.09*
O-30 t 0.01t
0.56 + 0.09t
of Ouabain VT i%kz
Ouabain, 1 pg/ kg min Ouabain, 1 pg/ kg-min, + C-l cord section Ouabain, 1 pgl kg min (stopped at 78 pg/kg) + C-l cord section
to
l
l
Values given are means SE for groups of 6 animals. * Sign% cantly different from neurally intact group receiving ou .abain (P < 0.01 1. t Significantly diffe rent from C-l cord section (P < 0.01) but not significantly different from neurally intact group. TABLE
ouabain
4. hfluence of spinal cord transection: infusion rate 2 M/kg- min Neurally
Dose of ouabain to VT, /%/kg Active Rb+ uptake at VT onset, nmol/mg wet wt 30 min Myocardial ouabain content at VT onset, pmol/mg wet wt
Intact
Significance
C-l
Cord
Section
94 Ik 5
NS
98 It 3
0.32 t 0.05
P < 0.025
0.19 z!I 0.01
0.59 ?I 0.05
P < 0.001
1.07 Ik 0.09
l
drug content and inhibition of monovalent cation active transport. In an additional set of experiments, ouabain was infused at 2 pg/kg min, twice the infusion rate studied above. At this rate of infusion, spinal section did not alter the cumulative dose required to produce ventricular tachycardia. However, in the neurally deprived (C-l sectioned) group, a higher myocardial ouabain content was present at onset of VT, and this was accompanied by significantly greater monovalent cation transport inhibition, to 30% of control at onset of VT, compared with neurally intact animals (Table 4). The arrhythmogenic dose of ouabain was 94 t 5 pg/kg in the neurally intact group and 98 t 3 rug/kg in the group with C-l cord section. However, active Rb+ transport was reduced from 0.32 t 0.05 nmol/mg 30 min in the neurally intact series to 0.19 t 0.01 nmol/ mg 30 min in the C-l cord section series, a 41% reduction, similar to the change of 44% observed in experiments employing a 1 pg/kg min ouabain infusion rate. l
l
l
l
DISCUSSION
Substantial evidence suggests that the sympathetic nervous system plays a part in the genesis of disorders of cardiac rhythm produced by digitalis glycosides (see reviews by Levitt et al. (17, 19) and Roberts et al. (26)]. Cardiac glycoside-induced augmented activity in sympathetic nerves leading to the heart has been reported by a number of workers (10, 11, 21). The surgical
RISLER,
AND
SMITH
interruption of this augmented activity by spinal cord transection at the atlantooccipital junction has been shown to provide protection against digitalis-induced arrhythmias in our study as well as in previous work (4, 8, 11, 18, 29). However, the protective effect of spinal .cord section has been difficult to interpret because of concomitant reduction of heart rate and arterial blood pressure. These effects have been shown to influence myocardial uptake of cardiac glycosides (2, 13, 20, 21). Previous studies have, therefore, utilized tritium-labeled digitalis preparations to determine if the rate of myocardial uptake was reduced in the spinal-sectioned animals and if a higher level of digitalis content in the myocardium was in fact required to produce arrhythmias when the myocardial sympathetic neural innervation had been interrupted (4, 18, 29). This study confirms that a higher myocardial content of digitalis is found in the neurally ablated group at the onset of ventricular arrhythmias. We further addressed the significance of the higher myocardial content in the neurally deprived preparation by determinating the degree of inhibition of monovalent cation transport associated with higher myocardial levels of ouabain. The potassium analog Rb+ is similar to K+ in its transport properties (3) and has been used as an indicator of Na+-K+-ATPase-dependent sodium and potassium transport in red blood cells (27, ZS), rat brain (3), guinea pig left atrium (6, 27), guinea pig ventricular slices (X5), and canine left ventricular samples (14). This study used the measurement of Rb+ uptake as an index of Na’-K+-ATPase-dependent monovalent cation transport in samples of cat ventricular myocardium. The results with left ventricular myocardial slices demonstrate linearity of Rb+ uptake beyond the time period studied (30 min). Furthermore, under the conditions of these experiments, active Rb+ uptake was quite similar to active K+ uptake (Table 1). Our results in this cat experimental model demonstrate a greater inhibition of monovalent cation active transport in left ventricular myocardium at the onset of overt ouabain cardiotoxicity in animals in which the sympathetic neural input to the heart is interrupted by C-l spinal cord section. These data indicate that the higher myocardial drug content found in the spinal preparation at onset of VT is biologically significant in that the drug has interacted specifically with Na+-K+ATPase, the putative receptor site for toxic effects of digitalis glycosides (1, 7, 12, 24, 25). The greater inhibition of Rb+ transport at onset of toxicity found in animals with spinal cord transection provides indirect support for the hypothesis that inhibition of Na+-K+-ATPase is related to the direct toxic effects of digitalis on the heart, although the present, data do not establish a causal relationship. With C-l cord section removing sympathetic neural influences on the heart, cardiac glycoside-induced ventricular arrhythmias emerge at a higher myocardial content and at greater levels of inhibition of active monovalent cation transport. The finding of greater transport inhibition by ouabain at the onset of sustained cardiotoxicity in cord-sectioned animals is consistent with the thesis that the sympathetic autonomic nervous system
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 16, 2019.
NEURAL
EFFECTS
OF DIGITALIS
H535
TOXICITY
facilitates the development of overt toxicity. This concept of arrhythmia facilitation is further supported by the observation that at similar levels of monovalent cation transport inhibition, neurally deprived animals are in sinus rhythm while neurally intact animals are in sustained ventricular tachycardia (Table 3). The similar cumulative ouabain doses to onset of VT with and without spinal section at the higher infusion rate of ouabain is in agreement with the previous report of Cagin et al. (5). The data presented here, however, show that the myocardial content of glycoside was higher and the inhibition of monovalent cation transport greater in the neurally deprived group, demonstrating a dependence of myocardial glycoside levels and monovalent cation active transport inhibition at onset of overt cardiotoxicity on the state of the nervous system. Thus the failure of cumulative doses per se at higher infusion rates to reflect changes in the mechanisms causing or facilitating cardiotoxicity could explain many of the past inconsistencies in the literature. The failure to find protection by antiadrenergic interin ventions in the past (23, 30, 31) may be explained part by this phenomenon. The determination of inhibition of myocardial monovalent cation active transport provides information over and above that obtained from myocardial cardiac glycoside levels, as exemplified by the data in Tables 3 and 4. The degree of Rb+ active uptake is essentially the same at onset of VT at the two rates of infusion in both spinal-sectioned and neurally intact animals,
whereas total dose and myocardial ouabain content show considerable variation. Thus the failure of cumulative dose during constant infusion to correlate with the onset of cardiotoxicity and its failure to reflect modifying influences of the nervous system can be reconciled in these experimental studies by the determination of active monovalent cation transport. These data provide further support for the concept that the intactness of the nervous system influences the response of the heart to a given dose of cardiac glycoside. Cardiotoxicity precipitated by ouabain involved an interaction of neurally mediated and direct cardiac actions of the glycoside in this experimental model. With the nervous system intact, bursts of nonuniform neural input appear to augment the direct cardiotoxic effects of digitalis (16), setting the stage for the development of potentially lethal cardiac arrhythmias. With the sympathetic neural input interrupted in spinalsectioned animals, higher myocardial ouabain content and greater inhibition of monovalent cation transport are observed at onset of ventricular arrhythmias, indicating that more receptor-bound drug is needed to produce overt cardiotoxicity. This investigation was supported by National Heart, Lung, and Blood Institute Fellowship lF32HL05542-01 and Grant HL-18003, by American Heart Association Award 76-597, and by the Paul-Martini Foundation. Received
24 April
1978; accepted
in final form 5 July 1978.
REFERENCES 1. AKERA, T., F. S. LARSEN, AND T. M. BRODY. The effect of ouabain on sodium and potassium activated adenosine triphosphate from the heart of several mammalian species. J. PharmacoZ. Exptl. Therap. 170: 17-26, 1969. 2. BELLER, G. A., T. W. SMITH, AND W. B. HOOD, JR. Altered distribution of tritiated digoxin in the infarcted canine left ventricle. CircuZation 46: 572-579, 1972. 3. BERNSTEIN, J. C., AND Y. ISRAEL. Active transport of Rbs” in human red cells and rat brain slices. J. Pharmacol. Exptl. Therap. 174: 323-329, 19’70. 4. CAGIN, N., J. C. SOMBERG, H. BOUNOUS, T. MITTAG, A. RAINES, AND B. LEVITT. The influence of spinal cord transection on the capacity of digitoxin to induce cardiotoxicity. Arch. Intern. Pharmacodyn. Therap. 207: 370-374, 1974. 5. CAGIN, N., J. C. SOMBERG, H. BOUNOUS, T. MITTAG, A. RAINES, AND B. LEVITT. Ouabain cardiotoxicity, a reassessment of methodology. Arch. Intern. Pharmacodyn. Therap. 224: 230-238,1976. 6. CURFMAN, G. D., T. J. CROWLEY, AND T. W. SMITH. Thyroidinduced alterations in myocardial sodium and potassium activated adenosine triphosphatase, monovalent cation active transport, and cardiac glycoside binding. J. CZin. Invest. 59: 586-590, 1977. 7. DUTTA, S., H. M. RHEE, AND B. H. MARKS. H”-ouabain accumulation and Na+, K+-ATPase activities in relation to therapeutic, toxic and lethal doses of ouabain in dogs. In: Recent Advances in Studies on Cardiac Structure and MetaboZism (Myocardial BioZogy), edited by N. S. Dhalla. Baltimore, MD: University Park Press, 1974, vol. 4, p. 119-130. 8. ERLIJ, D., AND R. MENDEZ. The modification of digitalis intoxication by excluding adrenergic influences on the heart. J. PharmacoZ. ExptZ. Therap. 144: 97-103, 19t,d. 9. FINK, B., AND A. SCHOOLMAN. Arterial blood acid base balance in unrestrained walking cats. Proc. Sot. Exptl. BioZ. Med. 112: 328-330, 1963. 10. GILLIS, R. A. Cardiac sympathetic nerve activity: changes induced bv ouabain and nronranolol. Science 166: 508-510. 1969.
11. GILLIS, R. ‘A.) A. RAINES, Y. J. SOHN, B. LEVITT, AND G. STANDAERT. Neuroexcitatory effects of digitalis and their role in the development of cardiac arrhythmias. J. PharmacoZ. Exptl. Therap. 183: 154-168, 1972. 12. GOLDSTEIN, R. E., S. C. PENZOTTI, K. S. KUEHL, K. H. PRINDLE, C. A. HALL, E. D. TITUS, AND S. E. EPSTEIN. Correlation of antiarrhythmic effects of diphenylhydantoin with digoxin induced changes in myocardial contractility, sodium potassium adenosine triphosphatase activity, and potassium efflux. CircuZation Res. 33: 175-182, 1973. 13. HOPKINS, B. E., R. R. TAYLOR, C. HENDERSON, AND P. BURROWS. Digoxin distribution in the dog’s left ventricle in the presence of coronary artery ligation. J. MOL. CeZZuZar CardioZ. 5: 197-203, 1973. 14. HOUGEN, T. J., AND T. W. SMITH. Inhibition of myocardial monovalent cation active transport by subtoxic doses of ouabain in the dog. Circulation Res. 42: 856-863, 1978. 15. Ku, D. D., T. AKERA, C. L. PEW, AND T. M. BRODY. Cardiac glycosides: correlations among Na+,K+-ATPase, sodium pump and contractility in the guinea pig heart. Arch. Exptl. PathoZ. PharmacoZ. 285: 185-200, 1974. 16. LATHERS, C. M., J. ROBERTS, AND G. J. KELLIHER. Correlation of ouabain-induced arrhythmia and nonuniformity in the histamine-induced discharge of cardiac sympathetic nerves. J. PharmacoZ. Exptl. Therap. 203: 467-479, 1977. 17. LEVITT, B., N. CAGIN, J. KLEID, J. SOMBERG, R. GILLIS. The role of the nervous system in the genesis of cardiac rhythm disorders. Am. J. CardioZ. 37: 1111-1113, 1976. 18. LEVITT, B., N. A. CAGIN, J. SOMBERG, H. BOUNOUS, T. MITTAG, AND A. RAINES. Alteration of the effects of distribution of ouabain by spinal cord transection in the cat. J. PharmacoZ. Exptl. Therap. 185: 24-28, 1973. 19. LEVITT, B., N. A. CAGIN, J. C. SOMBERG, AND J. KLEID. Neural basis of the genesis and control of digitalis arrhythmias. Cardiology 61: 50-60, 1976. - 20. LLOYD, B. L., AND R. R. TAYLOR. Augmentation of myocardial
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 16, 2019.
H536
21. 22.
23.
24. 25.
26.
digoxin concentration in hemorrhagic shock. CircuZation 51: 718-722, 1975. LLOYD, B. L., AND R. R. TAYLOR. Influence of myocardial mechanical activity and coronary blood flow on myocardial digoxin uptake. Cardiovascular Res. 10: 487-493, 1976. MCLAIN, P. Effects of cardiac glycoside on spontaneous efferent activity in vagus and sympathetic nerves of cats. Intern. J. Neuropharmacol. 8: 379-387, 1969. MORROW, D. H., T. E. GAFFNEY, AND E. BRAUNWALD. Studies on digitalis. VIII. Effect of autonomic innervation and of myocardial catecholamine stores upon the cardiac action of ouabain. J. PharmacoZ. ExptZ. Therap. 140: 236-242, 1963. OKITA, G. T. Dissociation of Na+,K+-ATPase inhibition from digitalis intropy. Federation Proc. 36: 22252230, 1977. RHEE, H. M., S. DUTTA, AND B. H. MARKS. Cardiac Na+,K+ATPase activity during positive inotropic and toxic actions of ouabain. European J. PharmacoZ. 37: 141-153,1976. ROBERTS, J., G. J. KELLIHER, AND C. M. LATHERS. Role of
SOMBERG,
27. 28.
29.
30. 31.
RISLER,
AND
SMITH
adrenergic influences in digitalis-induced ventricular arrhythmia. Life Sci. 18: 665-678, 1976. SMITH, T. W., AND H. WAGNER, JR. Effects of (Na+ + K+)ATPase specific antibodies on enzymatic activity and monovalent cation transport. J. Membrane BioZ. 25: 341-360, 1975. SMITH, T. W., H. WAGNER, JR., J. E. MARKIS, AND M. YOUNG. Studies on the localization of the cardiac glycoside receptor. J. CZin. Invest. 51: 1777-1789, 1972. SOMBERG, J. C., H. BOUNOUS, N. CAGIN, C. ANAGNOSTROPOULOS, AND B. LEVITT. The influence of prostaglandins E, and E, on ouabain cardiotoxicity in the cat. J. PharmacoZ. ExptZ. Therap. 203: 480-484, 1977. Digitalis WILLIAM, V. L., T. COOPER, AND C. R. HANLON. tolerance of the denervated heart (Abstract). Federation Proc. 24: 486, 1965. YELONSKY, J., AND R. ERVIN. The effect of ouabain on cardiac automaticity in reserpine-pretreated dogs. Am. Heart J. 62: 687689, 1961.
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 16, 2019.
