The strange behaviour of digitalis-like substances

How many digitalis-like substances?

More than 40 years ago, Hugh de Wardener postulated the existence of a natriuretic hormone that controls sodium homeostasis and which is secreted during sodium loading or volume-expanded states [¹]. Its action should have been mediated through the inhibition of the renal Na ⁺,K ⁺-pump [²]. If generalized to all cells of a mammalian organism, the concomitant inhibition of the Na ⁺,K ⁺-pump in vascular smooth muscle cells would have indeed increased the basal arteriolar tone, hence promoting hypertension [³]. Endogenous sodium pump ligands have subsequently been identified as having ‘digitalis-like’ activity and are commonly believed to facilitate natriuresis through inhibition of the Na ⁺,K ⁺-pump in renal tubular cells [⁴].

After solution of the methodological problems that shed doubt on the very existence of endogenous sodium pump ligands in mammals [⁵], it appears that at least two major kinds of digitalis-like sodium pump ligands can be found in the human circulation: the originally hypothesized endogenous cardenolide [ouabain-like factor (OLF), probably of hypothalamic and adrenal origin, an isomer of plant-derived ouabain] [^6–8] and a more recently identified molecular structure, the endogenous bufadienolide marinobufagenine (MBG), which is slightly different from cardenolides (structural analogs of proscillaridin A with a doubly unsaturated lactone ring), and is found in plasma and adrenals [^9,10]. Its name derives from the molecular structure being originally identified in amphibians. A detailed description of the chemico-physical characteristics of MBG and the structural differences from OLF have been described in detail elsewhere [^11,12].

To complicate the matter, there are at least three more compounds, mainly observed in volume-expanded states, that share the ability to inhibit renal Na ⁺ reabsorption but which do not have a digitalis-like structure: vanadium salts [¹³], an endogenous inhibitor of the renal Na-K-Cl cotransport [¹⁴] and another not yet clearly identified substance [¹⁵].

After accepting the existence of different inhibitors of the Na ⁺,K ⁺-pump in mammalian blood as proven, their pathophysiological action in particular districts through selective modulation of the Na ⁺,K ⁺-pump subunit isoforms starts to become clear. Also limiting our discussion on structural analogs of digitalis (MBG and OLF), experimental data indicate that they play distinct functional roles. MBG has greater affinity than ouabain for the α₁ subunit of the Na ⁺,K ⁺-ATPase, which is more abundant in vascular and renal tissue, whereas ouabain has greater affinity than MBG for the α₃ subunit, which is more abundant in neural sarcolemma and nerve endings [¹⁶]. Although structurally very similar, plant-derived ouabain and OLF have substantial differential affinities for the renal Na ⁺,K ⁺-pump. In fact, when tested in rats (a species whose renal Na ⁺,K ⁺-pump has a very low affinity for ouabain), both OLF obtained from rat or OLF obtained from bovine hypothalamus have three orders of magnitude greater affinity for renal Na ⁺,K ⁺-pump than ouabain [¹⁷]. This indicates that endogenous OLF should be able to bind the renal Na ⁺,K ⁺-pump in physiological concentrations.

It appears that both OLF and MBG have a biphasic action on the Na ⁺,K ⁺-pump. At low doses (in the picomolar range of physiological concentrations), they stimulate the Na ⁺,K ⁺-pump, whereas higher doses (one to two orders of magnitude compared to the physiological concentration) are required to inhibit it [^18–21].

Are all endogenous digitalis-like substances natriuretic?

Recently, Alexei Bagrov and colleagues have made great efforts to elucidate the role of MBG and OLF in Na ⁺ and volume-expanded states. In this issue of the journal, Fridman et al. measured MBG, OLF and α-hANP levels in a small number of patients with different severity of heart failure (from NYHA 1–4) and report that left ventricular diameter (both diastolic and systolic) as well as NYHA class correlated positively with MBG and α-hANP while ejection fraction correlated negatively [²²].

The finding of a significant positive correlation between MBG and α-hANP indicates that both MBG and α-hANP are secreted in a graded manner according to the severity of heart failure. Because MBG and α-hANP have opposite effects on the vascular tone despite a common natriuretic activity, the vasorelaxant action of α-hANP counteracts the vasoconstrictor action of MBG. No correlation with NYHA class or with ventricular performance is found with OLF. Their findings of no correlation of OLF level with the severity of cardiac failure is consistent with the lack of correlation of OLF with atrial filling pressure in another small sample of patients with different degrees of heart failure severity [²³], in spite that a significant negative correlation between OLF and cardiac index was found. How can we reconcile all these findings? An initial explanation may be found in the relative similarity in molecular structure between OLF and MBG. It is not known whether the experiments performed before MGB was described used antibodies that were specific enough to efficiently discriminate between the two digitalis-like factors. Consequently, considering only the more recent data, we can try to provide some alternative explanations.

MBG increases in parallel with blood pressure both in salt-sensitive and in salt-resistant Dahl rats under chronic Na ⁺ load, whereas OLF increases sharply but only during the first week of Na ⁺ load. The experiment has been performed in rats fed 8% Na ⁺ diet for 4 weeks. Pretreatment with anti-OLF or anti-MBG antibodies reduced the excretion of a Na ⁺ load by the same extent in non-salt-loaded salt-sensitive Dahl rats [²⁴] suggesting that both substances should have natriuretic activity. The picture is different in MHS, a less severe model of rat hypertension. Two weeks of Na ⁺ loading did not change OLF concentration, whereas 5 months of a very low Na ⁺ diet (0.1%) doubled plasma OLF in the hypertensive MHS strain (P. Ferrari, personal communication). In addition, in man, there are no data indicating that OLF has direct natriuretic activity, nor that it is produced in larger amounts in volume-expanded states. Conversely, OLF increases after acute and chronic Na ⁺ depletion [²⁵]. This observation, together with the biphasic action on of OLF on the Na ⁺,K ⁺-pump, suggests that OLF (and possibly MBG) acts as an anti-natriuretic substance in humans, at least under physiological conditions.

Support for this hypothesis is also found in the study by Fridman et al. [²²], where the correlation between α-hANP and MBG does not necessarily mean that MBG is natriuretic as well as α-hANP, but that a digitalis-like substance secretion may be stimulated by α-hANP, as previously demonstrated [^26,27]. To solve this problem, it would have been interesting to study ANP secretion during acute saline infusion and pretreatment with anti-MBG or anti-OLF antibodies in earlier experiments [²⁴]. The same experiment provides further evidence that at least OLF is not natriuretic. In fact, the reduction of Na ⁺ excretion after pretreatment with anti-MBG antibodies also reduces OLF excretion, again as if OLF production were downregulated by Na ⁺ retention induced by anti-MBG antibody pretreatment.

Whatever the case, high levels of ouabain-like factors (either OLF or MBG) may be the mediator of sympathetic overactivity observed in heart failure [²⁸], which is a major contribution to the worsening of heart failure itself.