Endogenous Digitalis-like Substance Research Articles

Clinical Analysis of Hyponatremia in Acute Craniocerebral Injury

To explore pathological mechanisms of central hyponatremia and its treatment. Synchronous assay was made for changes of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), endogenous digitalis-like substance (EDLS), antidiuretic hormone (ADH) in blood, and Na(+) concentrations in blood and urine, and plasma- and urine-osmolality in 68 patients with acute craniocerebral injury (ACI). Of the 68 patients with ACI, 27 were found to have hyponatremia, and such illness was mostly concentrated on severe cases. The central hyponatremia in patients with ACI may be related to the increase in the secretion of EDLS and ADH as the result of damaged functions of the hypothalamic-hypophysial system, and it seems that the decrease in blood ANP and BNP has no direct effect on Na(+) concentrations in blood. Inappropriate secretion of antidiuretic hormone syndrome and cerebral salt-wasting syndrome are the two main reasons for hyponatremia in patients with craniocerebral injury. The pathological mechanism, diagnostic standards, as well as treatment methods for the two, however, are not just the same. Intravenous injection of extrinsic thyrotropin-releasing hormone might inhibit dilutional hyponatremia arising from the increase in ADH secretion by patients with ACI.

Natriuretic Peptides, Antidiuretic Hormone and Hyponatraemia after Acute Craniocerebral Injury

We investigated the physiological mechanisms involved in central hyponatraemia in patients with acute craniocerebral injury (ACI). We measured blood concentrations of natriuretic peptides, antidiuretic hormone (ADH), and endogenous digitalis-like substance (EDLS), blood and urine sodium concentrations, and the plasma and urine osmolality in 68 patients with ACI and 24 healthy control subjects. A total of 27 ACI patients were hyponatraemic and the majority of these had grievous or severely grievous craniocerebral injuries. Blood concentrations of EDLS and ADH in hyponatraemic ACI patients were significantly higher compared with normonatraemic ACI patients and control subjects. Blood EDLS and sodium concentrations were negatively correlated with each other, whereas EDLS was positively correlated with urine sodium concentration and with urine osmotic pressure. Hyponatraemic ACI patients require different treatment based on the cause of their central hyponatraemia, so it is important to undertake a comprehensive analysis of each patient's physiological status.

MECHANISM OF ANTIHYPERTENSIVE EFFECT OF DIETARY POTASSIUM IN EXPERIMENTAL VOLUME EXPANDED HYPERTENSION IN RATS

Dietary potassium supplementation lowers blood pressure (BP) and attenuates complications in hypertensive subjects, particularly those with the low renin volume expanded (LRVE) variety. We and others have shown that the plasma level of a digitalis like substance (DLS) is elevated in this type of hypertension. We therefore, examined the effect of increases in dietary potassium on the plasma level of endogenous DLS, myocardial and renal Na+, K+-ATPase (NKA) activities, BP, and renal excretory function in reduced renal mass (RRM)-salt hypertension in the rat, a classical model of LRVE hypertension. 70% RRM rats were divided in 4 groups, namely those consuming: 1) a sodium free and normal potassium (1.3% as KCl) diet (RRM-0 Na), 2) a normal sodium and normal potassium diet (RRM-NaK), 3) a normal sodium and high potassium (2 X normal) diet (RRM-Na2K), and 4) a normal sodium and 4 times normal potassium diet (RRM-Na4K). At the end of 4 weeks of dietary treatment, direct BP was recorded, plasma level of DLS determined by bioassay and with a radioimmunoassay for digoxin (DIF) and myocardial and renal NKA activities were measured. As expected, compared to RRM-0Na rats, RRM-NaK rats developed hypertension. BP increased significantly less in RRM-Na2K, whereas BP did not increase in RRM-Na4K rats. Hypertension in RRM-NaK rats was associated with an increase in plasma DLS and DIF and decrease in renal and myocardial NKA activities. DLS was increased (DIF was not changed) and myocardial NKA also decreased in rats consuming double potassium. However, quadrupling potassium in the diet (RRM-Na4K) normalized DLS and DIF and increased myocardial and renal NKA activities, compared to RRM-0Na rats. Also compared to RRM-0Na, water consumption, urinary volume excretion, sodium, and potassium increased in the other 3 groups, more so in RRM-Na4K rats. These data show that quadrupling the potassium in the diet prevents the BP increase in RRM rats and this is associated with diuresis/natriuresis and normalization of DLS, perhaps because the diuresis/natriuresis normalizes blood volume.

Effect of administration of insulin on streptozotocin-induced diabetic hypertension in rat.

We have reported that streptozotocin-induced insulin-dependent diabetes mellitus in 25% reduced renal mass rats is associated with low-renin, volume-expanded hypertension and that the development of the hypertension can be prevented with insulin. In this study we examined the effect of insulin after the animals had developed sustained hypertension. Normotensive 25% reduced renal mass rats were treated with streptozotocin and, as expected, developed insulin-dependent diabetes mellitus and hypertension. After 4 weeks of sustained hypertension, neutral protamine Hagedorn insulin (6 to 8 IU/d) was administered subcutaneously for 4 weeks. As expected, insulin treatment decreased plasma glucose and increased body weight gain relative to untreated diabetic rats. On the other hand, insulin treatment did not reverse the hypertension and albuminuria. It also did not normalize extracellular fluid volume and plasma renin activity. Furthermore, insulin treatment did not reverse the increase in plasma Na+,K(+)-ATPase inhibitory activity (determined by both radioimmunoassay and bioassay) and the inhibition of myocardial microsomal Na+,K(+)-ATPase activity observed in the untreated diabetic hypertensive rats. 5'-Nucleotidase, a membrane marker, was not different between insulin-treated and untreated diabetic rats. These results show that insulin, given as here described, does not reverse the insulin-dependent diabetes mellitus hypertension in 25% reduced renal mass rats once it is established, perhaps because it does not reverse the albuminuria, volume expansion, increase in endogenous digitalis-like substance, and inhibition of cardiovascular muscle cell Na+,K(+)-ATPase activity.

Role of digitalis-like substance in the hypertension of streptozotocin-induced diabetes in reduced renal mass rats.

We have previously reported that chronic hypertension develops consistently in Wistar rats with a 25% reduction in renal mass (RRM) following the induction of insulin dependent diabetes mellitus (IDDM) with streptozotocin (STZ, 65 mg/kg body weight, intravenously). In this study, we examined the role of the endogenous digitalis-like substance in the development of hypertension. Four groups of rats were studied: 1) 25% RRM rats with STZ-induced IDDM (25-DM), 2) normal rats with STZ-induced IDDM (2K-DM), 3) 25% RRM rats with vehicle treatment (25-V), and 4) normal rats with vehicle treatment (2K-V). In 25-DM rats, blood pressure progressively increased during the 3 weeks after STZ treatment and was associated with microalbuminuria, low plasma renin activity, and extracellular volume expansion. In contrast, the 2K-DM, 25-V, and 2K-V rats remained normotensive. Furthermore, the plasma and urine levels of digoxin-like immunoreactive factor (DIF), determined by digoxin radioimmunoassay (Baxter), were significantly higher in hypertensive 25-DM rats than in their controls. The same was the case for plasma digitalis-like substance (DLS), determined by exposing canine Na+,K(+)-ATPase to plasma fractions and observing the percent inhibition. Increased DIF and DLS in hypertensive 25-DM rats was associated with a significant decrease in Na+,K(+)-ATPase activity of microsomes prepared from the left and right ventricles, when compared with microsomes from normotensive 2K-DM animals. Microsomal 5'-nucleotidase, a plasma membrane marker, was unchanged. The DIF and DLS correlated significantly with each other and with myocardial Na+,K(+)-ATPase activity and mean blood pressure. These results suggest that increased endogenous digitalis-like substance, which inhibits cardiovascular muscle cell Na(+)-K(+)-pump activity, may be involved in the mechanism of hypertension associated with IDDM in 25% RRM rats.

Role of digitalis-like substance in experimental insulin-dependent diabetes mellitus hypertension.

Hypertension is frequently associated with insulin-dependent diabetes mellitus, but the mechanism of the hypertension is unknown. An animal model of insulin-dependent diabetes mellitus hypertension could be helpful in determining the mechanism, but experimental insulin-dependent diabetes mellitus has been infrequently and irregularly associated with hypertension. In an attempt to develop a dependable model of insulin-dependent diabetes mellitus hypertension, we studied seven series of rats receiving either streptozotocin, surgical reduction of renal mass, or both. We found that superimposing streptozotocin 65 mg/kg body weight on 25% reduced renal mass regularly produced insulin-dependent diabetes mellitus and low-renin volume-expanded hypertension and that the animals remained healthy and hypertensive for as long as followed (13 weeks). Microalbuminuria correlated temporally with blood pressure. We used this dependable model to examine the role of endogenous digitalis-like substance in the development of hypertension in insulin-dependent diabetes mellitus. Plasma levels of digoxin-like immunoreactive factor (DIF), determined with a digoxin radioimmunoassay, were significantly higher in these hypertensive rats than in normotensive control rats (two-kidney diabetic rats, 25% reduced renal mass rats receiving vehicle for streptozotocin). This increase in plasma DIF was associated with a decrease in Na+, K(+)-ATPase activity in microsomes prepared from left or right ventricle. Microsomal 5'-nucleotidase, a plasma membrane marker, was unchanged. The plasma DIF level correlated inversely with myocardial Na+, K(+)-ATPase activity and positively with systolic blood pressure.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of the endogenous digitalis-like substance (EDLS)-containing neurons labeled by digoxin antibody in hypothalamus and three circumventricular organs of dog and macaque

Endogenous digitalis-like substance (EDLS) is a newly discovered humoral agent which causes sodium-diuresis. EDLS is well known to have inhibitory activity to Na +, K +-ATPase and cross-immunoreactivity to digoxin antibody; however, its precise chemical structure has not yet been determined. We had previously developed a histochemical technique for EDLS, i.e., digoxin-immunohistochemistry, and demonstrated that EDLS was produced in the hypothalamic neurons. In the present study, the distribution of EDLS-containing neurons in the hypothalamus of dog and macaque was investigated using this technique, because anti-EDLS antibody cannot be obtained yet. In both species, EDLS neuronal somata were mainly localized in the paraventricular nucleus and the supraoptic nucleus and its accessory nuclei. A number of somata were also scattered in the other hypothalamic areas. The processes of these neurons ran from the area where the somata were located, through the lateral and basal area of the hypothalamus, to the infundibulum. These nerve fibers with varicosities were associated with the primary capillaries of hypophysial portal veins. A few immunopositive nerve fibers were also seen in the pituitary posterior lobe of both species. Intensive immunoreactivities were observed in the subfornical organ and organum vasculosum laminae terminalis. There were no differences between dog and macaque.

Histological study on ouabain immunoreactivities in the mammalian hypothalamus

The endogenous degitalis-like factor (EDLF) which has recently been purified from human plasma and identified as ‘ouabain’, a cis-trans-cis steroid of plant origin, is thought to be similar to the hypothetical humoral factor, ‘endogenous digitalis-like substance (EDLS)’. In order to examine the hypothesis that EDLS is produced in the hypothalamus, we prepared an ouabian-specific antibody, and applied it to rat and macaque brains. Ouabain immunoreactivities were observed in the hypothalamus of both species. The immunopositive neurons were distributed in paraventricular and supraoptic nuclei, and some other hypothalamic regions. Their nerve fibers were seen abundantly in the hypothalamo-neurohypophysial regions. These results strongly support the possibility of existence of cis-trans-cis steroid including EDLF in mammalian brain.

Reduction of erythrocyte (Na +-K +)ATPase activities in non-insulin-dependent diabetic patients with hyperkalemia

To elucidate the mechanism of hyperkalemia in diabetic patients without renal failure, we investigated (Na +-K +) adenosine triphosphatase (ATPase) activity in erythrocyte membrane, erythrocyte Na + and K + content, and plasma endogenous digitalis-like substance in control subjects (n = 16) and non-insulin-dependent diabetes mellitus (NIDDM) patients (n = 62). NIDDM patients were divided into normokalemic patients (NKDM, n = 48) and hyperkalemic patients (HKDM, n = 14). There was no difference in plasma glucose or hemoglobin A 1c (HbA 1c) levels, plasma renin activity (PRA), and plasma aldosterone concentrations (PAC) between NKDM and HKDM patients. (Na +-K +)ATPase activities in NIDDM patients were significantly reduced compared with those in control subjects (0.336 ± 0.016 μmol-inorganic phosphate [Pi]/mg protein/h, mean ± SEM, P < .05), and (Na +-K +)ATPase activities in HKDM patients (0.243 ± 0.015 μmol Pi/mg protein/h) were significantly reduced compared with those in NKDM patients (0.295 ± 0.008 μmol Pi/mg protein/h, P < .01). Plasma K + content had a significant negative correlation with (Na +-K +)ATPase activity in diabetic patients ( r = −.365, P < .01). Erythrocyte Na + content had a significant negative correlation with (Na +-K +)ATPase activity in control subjects ( r = −.619, P < .05). There was no difference in plasma endogenous digitalis-like substance among the three groups. (Na +-K +)ATPase activity was not significantly correlated with plasma endogenous digitalis-like substance in control subjects and diabetic patients. These findings suggest that the reduction of (Na +-K +)ATPase activity, which was not related to plasma digitalis-like substance, may be partly responsible for hyperkalemia in diabetic patients.

Mild exercise decreases plasma endogenous digitalislike substance in hypertensive individuals.

Changes in a plasma endogenous digitalislike substance were investigated in relation to the antihypertensive mechanism of mild exercise. Fifteen women with mild essential hypertension and seven normotensive female volunteers were divided into exercised hypertensive (n = 10), nonexercised hypertensive (n = 5), and nonexercised normotensive (n = 7) groups. A 4-week general clinical observation period preceded the study period of 10 weeks. The exercised hypertensive individuals were treated with a lactate threshold exercise that corresponded to approximately 50% of the maximum oxygen consumption three times a week, whereas the nonexercised groups were observed at the outpatient clinic as control groups. In the exercised group, systolic blood pressure fell by 7 mm Hg (p = 0.05), diastolic by 6 mm Hg (p less than 0.01), and mean blood pressure by 7 mm Hg (p less than 0.01) after 10 weeks. The reduction in the plasma endogenous digitalislike substance was significant after 7 (-1.02 ng/ml, p less than 0.05) and 10 (-1.04 ng/ml, p less than 0.05) weeks in this group. It positively correlated with the reduction in diastolic (r = 0.70, p less than 0.05) or mean (r = 0.66, p less than 0.05) blood pressure and with changes in plasma norepinephrine (r = 0.76, p less than 0.05). The mean corpuscular volume of erythrocytes decreased (-1.7 fl, p less than 0.01) after 10 weeks of exercise, and the plasma volume index tended to decrease (-108 ml/m2, p = 0.28). In the control groups, significant changes in blood pressure and plasma endogenous digitalislike substance were not observed.(ABSTRACT TRUNCATED AT 250 WORDS)

DIGOXIN-LIKE IMMUNOREACTIVITY INCREASES WITH INTRAVENOUS INFUSION OF SALINE IN RATS

1. The effects of sodium-loading on releases of endogenous digitalis-like substance were investigated by measuring digoxin-like immunoreactivity (DLI) during intravenous infusions of isotonic (0.15 mol/L) and hypertonic (1 mol/L) saline in anaesthetized rats. Plasma DLI was measured after death by a digoxin-radioimmunoassay. 2. The infusion of isotonic saline and hypertonic saline elevated the central venous pressure to similar levels. The plasma DLI concentration in both the infused groups rose significantly compared with that in the control rat not receiving the intravenous infusion. 3. The difference in the hypothalamic concentrations of DLI was not significant among the three groups, however, there was a significant inverse relationship between the plasma and hypothalamic concentrations of DLI. 4. Results indicate that the central venous pressure, but not sodium concentration, is essentially involved in the release of DLI, possibly from the hypothalamus.

Elevated endogenous digitalis-like substance in hypertensive diabetic patients with a family history of hypertension

Endogenous digital-like substance (DLS) is increased in patients with essential hypertension and is hypothesized to play a role in the pathogenesis of high blood pressure. Whether an increase in DLS in diabetic patients with hypertension is associated with a family history of hypertension or diabetic nephropathy was investigated. Plasma DLS was measured as Na(+)-K(+)-ATPase inhibitory activity (ATPI) in 100 Type 2 diabetic patients. Ouabain was used as a standard of Na-K-ATPase inhibition. Diabetic patients with hypertension demonstrated a greater ATPI level than normotensive diabetic patients (p less than 0.05). In patients with hypertension groups, the positive family history group had a higher ATPI level than the negative family history group (p less than 0.01). Microalbuminuria was not correlated with the ATPI level in diabetic patients. These results suggest that ATPI might play a role in the pathogenesis of hereditary hypertension associated with diabetes mellitus, but not have etiologic significance in diabetic nephropathy.

Effects of bufalin on renal venous outflow, urine flow and natriuresis in the anesthetized dog.

Recent studies suggest that the endogenous digitalis-like substance (DLS) implicated in the pathogenesis of low renin hypertension in animals and humans may be a steroidal dienolide derivative. One of these derivatives, bufalin (aglycone), inhibits Na+,K+-ATPase (1) and has recently been shown in our laboratory to have some of the physiological characteristics expected of a DLS (2). For example, infusion of bufalin into the brachial artery of the anesthetized dog increases vascular resistance and blocks K+ vasodilation while it potentiates norepinephrine vasoconstriction (Fig. 1). When bufalin is administered intravenously in the dog, increases in heart rate, dP/dt, and arterial blood pressure ensue (Fig. 2). In some animals (3/7) post-infusion natriuresis and diuresis occurs. Intravenous infusion in the rat likewise produces increases in blood pressure, heart rate, and dP/dt, and marked diuresis, natriuresis and kaliuresis (3) during and post-infusion. The present study in the anesthetized dog has been designed to examine the mechanism for the post-infusion natriuresis and diuresis seen with bufalin.

The Transient Increase of Urinary Digitalis-like Substance Excreted During Excess Sodium Intake in Reduced Renal Mass Rats

Urinary immunoreactive endogenous digitalis-like substance (EDLS) excretion was studied in gradually reduced renal mass rats (RRM). Urinary EDLS increased immediately after the start of 1% NaCl ingestion, then it returned to the basal level 2 weeks later. Both urinary sodium excretion and urinary EDLS were significantly higher in 3/6 and 4/6 RRM than in control until 2 weeks after starting 1% NaCl. However, there was no difference in blood pressure between the groups. Transient EDLS increase may play an important role in maintaining sodium and water homeostasis, but its transient increase apparently does not contribute to blood pressure elevation.

Hemodynamic effects of bufalin in the anesthetized dog.

Studies in Lichstein's laboratory suggest that the endogenous digitalislike substance implicated in low renin hypertension might be a steroidal dienolide derivative. If this is true, the bufadienolides should block potassium vasodilation and enhance norepinephrine vasoconstriction, constrict blood vessels, raise blood pressure, and produce natriuresis and diuresis. We have therefore examined these parameters while infusing bufalin (aglycone), a bufadienolide, intrabrachially and intravenously in the anesthetized dog. Intrabrachial infusion of 5-25 micrograms/min with brachial arterial blood flow held constant at 100 ml/min produced a dose-dependent increase in perfusion pressure with rapid onset and offset, a progressive decrease in the vasodilator response to intrabrachial injection of 1 ml iso-osmotic potassium chloride solution (but not to acetylcholine), and an increase in the vasoconstrictor response to intrabrachial injection of 0.1 microgram norepinephrine. Intravenous infusion at 5-50 micrograms/min produced a dose-dependent increase in systemic arterial blood pressure, rate of change of ventricular pressure (dP/dt), and after the highest dose, cardiac irregularities. Natriuresis and diuresis were not observed. Thus, bufalin does in fact have some of the physiological properties required to be considered a candidate for the digitalislike substance found in low renin hypertension.

The Possible Role of Endogenous Digitalis-Like Substance in the Regulation of Circadian Changes in Urinary Electrolyte Excretion in Man.

The urinary volume (U.V.), Na excretion (UNaV) and K excretion (UKV) have been reported to show a circadian rhythm in man, but the mechanism of this rhythm has not been made clear. To investigate how atrial natriuretic peptide (ANP) and endogenous digitalis-like substance (DLS) participate in the circadian change in urinary electrolyte, the circadian changes in ANP and DLS (digoxin-like immunoactivity: DLI, Na-K-ATPase inhibitor: ATPI, ouabain binding inhibitor to Na-K-ATPase: OBI) were evaluated in 5 normal man. ANP, DLI and OBI showed no significant correlation with urinary electrolyte excretion, but there was a significant positive correlation between plasma ATPI and urinary Na excretion. From these results it is suggested that circulating Na-K-ATPase inhibitor (plasma ATPI) may be involved in the regulation of the circadian rhythm of urinary Na excretion.

Correlations between plasma levels of an endogenous digitalis-like substance and haemodynamic parameters measured during cardiac catheterization.

It has been postulated that one or more plasma digitalis-like compounds may play an important role in body fluid regulation and in essential hypertension, although very little is known about their possible role in general haemodynamics. We therefore measured plasma inhibition of human kidney Na+,K+-ATPase and plasma cross-reactivity with digoxin antibodies in 11 normotensive cardiopathic subjects admitted to our clinic for heart catheterization. Possible correlations with haemodynamic parameters were studied. Plasma digoxin-like activity correlated directly with left atrial pressure and with pulmonary circulation data. The ability of the plasma to inhibit Na+,K+-ATPase showed an inverse correlation with cardiac output and cardiac index. No correlations were found with any of the other parameters measured, notably systemic resistance, blood pressure and natriuresis. These findings suggest the presence of more than one substance sharing chemical properties with digitalis: (1) a substance cross-reacting with digoxin antibodies and dependent on pulmonary vascular congestion; and (2) a substance capable of inhibiting the Na+-K+ pump and present in large amounts in heart diseases with a reduced cardiac index.

Digitalis-like substance is produced in the hypothalamus but not in the adrenal gland in rats.

We studied the role of the adrenals on the plasma levels and urinary output of the digoxin-like immunoreactivity in order to elucidate the interrelationship between the adrenals and hypothalamic digoxin-like immunoreactivity. Urine was collected for 24 h 6 days after the bilateral adrenalectomy and then rats were killed by decapitation. Urinary excretion of digoxin-like immunoreactivity did not differ significantly between the sham-operated and adrenalectomized groups, regardless of sodium intake. Plasma levels did not change significantly with sodium-loading, and adrenalectomy did not significantly affect the plasma concentrations of the immunoreactivity. However, bilateral adrenalectomy increased the content of digoxin-like immunoreactivity in the hypothalamus significantly in both rat groups fed with a regular (P less than 0.05) and a high-salt (P less than 0.02) diet. However, when the levels were measured only 16 h after bilateral adrenalectomy, both plasma and hypothalamic contents were significantly higher in the high-salt than the regular-salt group. The correlation between the plasma and hypothalamic content was significant (P less than 0.01). These results strongly suggest that digoxin-like immunoreactivity is produced in the hypothalamus but not in the adrenals, and that the adrenal glands influence the turnover of the hypothalamic endogenous digitalis-like substance.

Endogenous digitalislike substance in an adult population in Japan.

Possible involvement of an endogenous digitalislike substance (EDLS) in blood pressure regulation was investigated using a Japanese population. Mean arterial pressure (MAP) significantly correlated with urinary excretion of the EDLS, age, and the obesity index. The plasma EDLS correlated with urinary EDLS. Urinary EDLS excretion well correlated with the inhibitory activity on Na+,K+-ATPase, and also with the urinary excretion of NaCl. Obesity index correlated with the Na+,K+-ATPase inhibition and arterial pressure. Although plasma content of atrial natriuretic polypeptide correlated with the urinary Na+,K+-ATPase inhibition, it did not correlate with the rest of all parameters. Plasma vasopressin level did not correlate with these parameters either. These results clearly indicate that the circulating EDLS (ie, Na+,K+-ATPase inhibitor) is implicated in the hypertension associated with an excess intake of sodium, aging and obesity.

Immunohistochemical studies on the distribution of endogenous digitalis-like substance(EDLS)-containing neurons in the rat hypothalamus, with special consideration on the possibility of their coexistence with posterior lobe hormones.

The distribution of the perikarya of endogenous digitalis-like substance (EDLS)-containing neurons in the rat hypothalamus was studied by immunohistochemistry using digoxin and digitoxin antiserum. In addition, the possible coexistence of EDLS and posterior lobe hormones was examined using an immunohistochemical double-staining technique. Digoxin-like immunoreactive neurons were demonstrated in the paraventricular and supraoptic nuclei and other hypothalamic areas. However, digitoxin-like immunoreactive neurons were not detected in the hypothalamus. A portion of the digoxin-like immunoreactive neurons showed immunoreactivity for vasopressin or oxytocin. From these results, it is suggested that these digoxin-like immunoreactive neurons correspond to "EDLS-producing neurons," and that EDLS seems to act not only as a natriuretic substance but also as a neurohormone or neurotransmitter.