Binding of catecholamines to phospholipids isolated from adrenal medullary granules.

The large granules of the adrenal medullary fraction were lysed and subjected to Sepharose 2B gel filtration. Proteophospholipids (PPL) were isolated from the membrane fraction and used for binding of catecholamines (CA), such as noradrenaline (NA), adrenaline (A) and dopamine (DA). The affinity order was DA greater than NA = A. The binding capacity was dependent on amine concentration, pH and ionic strength, and amounted to about 2 and 3.5 mumol per mumol lipid phosphorus in ether and ether-ethanol, respectively. A Scatchard plot revealed only one type of binding site; Kd = 8.3 mM. The anionic detergent sodium dodesylsulphonate (SDS) moderately increased the uptake of NA and A while cationic detergents like cetyltrimethylammonium bromide (CTAB) and Hyamine 2389 strongly diminished the binding to PPL. The ionic detergent sodium deoxycholate (DOC) and the non-ionic detergent Triton X-100 were without significant effect. It is suggested that the dipolar head group of the granule lipids, particularly the lipid phosphate groups, are involved in electrostatic or complex interactions with the CA, either directly, or through water molecules within the lipid polar head group.

Incorporation of biogenic amines into phospholipids studied in two-phase-system.

Phospholipids (PL) from splenic nerves, splenic nerve granules, adrenal medullary granules and egg yolk were used as binding ligands for studies on uptake of various biogenic amines. Efficient uptake of catecholamine (CA) occurred at low ionic strength (about 2-5 mM) and at pH 6.8-7.7. This uptake was also a function of the amine concentration in the aqueous medium. At higher ionic strength (e.g. 128 mM K-phosphate buffer) an uptake still occurred, but to considerably less extent, apparently since the association/dissociation ability of the amines within the PL was a function of cation concentration in the aqueous media. Sucrose- and dextrose-induced increase in the osmolarity of the aqueous medium did not to any appreciable extent reduce the CA uptake. Scatchard plot indicated that only one type of binding sites was engaged when PL from splenic nerves was used. Using PL from splenic nerves the following affinity order was obtained for the amine tested: 5-HT greater than TA greater than DA greater than NA greater than IP greater than A. When PL from medullary granules was used the order was: DA greater than IP greater than A greater than or equal to NA. No or negligible uptake of Dopa and its metabolites Doma and Dopac occurred. When added to the aqueous medium, the cationic amphiphilic detergent CTAB induced some uptake of Doma and Dopac, but no a Dopa. However, CTAB considerably diminished the uptake of NA and the other amines. On the other hand, ionic (DOC) and anionic (SDS) detergents strongly increased the uptake of NA and other amines into PL. The nonionic Triton X-100 was without significant effect on uptake of NA. It is suggested that the binding ligands may be present in the dipolar head of the PL molecules, particularly in the lipid phosphate group. The lipid protein may also be partially involved since chromatographic experiments showed that the CA accompanied lipid phosphorus and lipid protein. Electrostatic or complex interaction of CA within the polar head group or in conjunction with lipid protein would constitute an amine storage with efficient association/dissociation capacity.

Separation and purification of blood plasma arginine vasopressin on Sephadex G-50M for subsequent radioimmunoassay.

A gel filtration method has been developed for purification and separation of plasma arginine vasopressine (AVP) from proteins interfering with RIA-determination of AVP. The method involves filtration of 1 ml of plasma on a Sephadex G-50M gel column and the eluant used is 100 mM borate buffer. This rapid, and relatively simple method gives rather complete separation of plasma protein and AVP with an AVP recovery of about 90% at normal or elevated plasma AVP levels. Since 30 plasma samples may be run simultaneously, the method has potential value for routine laboratory use.

Cerebral sodium/angiotensin interaction studied by RIA-determination of urinary arginine vasopressin in the hydrated goat.

Radioimmunoassay determination of urinary arginine vasopressin (AVP) was employed to study quantitatively cerebral Na+/angiotensin II (A II) interaction in the hydrated goat. The solutions infused for 30 min at 0.02 ml/min into the lateral cerebral ventricle were: a) Hypertonic (0.25 M) NaCl, b) AII (0.3 ng/kg min) in isotonic (0.15 M) NaCl, and c) A II (doses as in b) in 0.25 M NaCl. The mean amounts of AVP detected in the urine in response to the various infusions were: a) 2.8 ng, b) 3.6 ng, and c) 13.3 ng. Thus, the A II/NaCl stimulation induced a detected renal excretion of AVP that was two times as large as the sum of the effects recorded in response to separate stimuli. Infusion c) invariably induced a pronounced, long-lasting inhibition of the water diuresis, intense thirst, and natriuresis. The corresponding effects of infusions a) and b) were much weaker and, as regards thirst and natriuresis, inconsistent. The determinations of renal AVP excretion provide additional, and rather direct evidence for the concept of a synergistic action of elevated cerebrospinal fluid [Na+] and A II as concerns cerebral control of fluid balance. With regard to this kind of interaction, the observed dipsogenic and natriuretic effects mainly confirm earlier observations.

Deuterium induced extinction of ADH-release in response to intracerebroventricular infusions of hypertonic NaCl and angiotensin.

Infusions (20 microliter/min) of hypertonic (0.3 M) NaCl and angiotensin II (1 ng/kg min-1) in isotonic (0.15 M) NaCl were made for 1 h in the hydrated goat during fully developed water diruesis. Either H2O or deuterium (D2O) WAS USED AS SOLVENT. A pronounced antidiuretic response, outlasting the infusion period by 30 min or more, was seen when the substances were dissolved in H2O. Only a weak inhibition of the water diuresis, which was extinguished during the infusion period, was obtained when D2O was used as the solvent. The infusion of 0.3 M NaCl/H2O invariably induced drinking in one of the goats, which, however, showed no drinking response to the infusions of 0.3 M NaCl/D2O. The possibility is discussed that D2O (perhaps by its inhibitory effect on (Na+-K+)-ATPase activity) reduced the sensitivity of juxtaventricular receptors regulating ADH-release and water intake.

Perturbations in fluid balance induced by medially placed forebrain lesions.

Acute and chronic effects on the fluid balance of radio-frequency forebrain lesions were studied in the goat. Medial lesions which involved practically the entire anterior wall of the third cerebral ventricle cause persistent loss of thirst and lack of significant antidiuretic hormone (ADH) release in response to hypernatraemia and plasma hyperosmolality. As acute response to such lesions an uncompensated, temporary water diuresis was seen, which rapidly caused pronounced hypernatraemia and hypovolaemia. Lesions extending laterally to encroach upon the supraoptic nuclei resulted in persistent signs of weak, inappropriate ADH secretion (=impaired water diuresis, renal salt wasting, and pronounced hyponatraemia during hydration). Forebrain damage, mainly restricted to the septal region, caused hyperdipsia. In some goats, obvious post-lesioning increase in salt appetite was observed which could not be coreelated to the extent of their forebrain damage. The results are discussed in relation to hypothalamic syndromes in man and previous studies on central control of fluid balance in the goat.

Recovery of ADH activity in the urine of goats under normal and stimulated conditions.

The hydrated goat was used for bioassays of antidiuretic hormone (ADH) activity recovered by resin column separation from the urine of other animals of the same species. A methodological innovation in the separation procedure was ethanol purification of the column before elution. In this manner substances which sometimes obscured the bioassays were eliminated without loss of ADH activity. With the bioassay method employed, it was possible to determine to the nearest 0.5 of a mU the ADH activity of unknown samples, provided they contained between 0-5 mU of ADH. When arginine vasopressin was infused intravenously into hydrated animals, slightly more than 10% of its antidiuretic activity was recovered in the urine. In the water replete goat the ADH activity found in the urine was of the order of 1 mU per hr urine secretion, indicating a neurohypophyseal ADH release of approximately 5 muU/kg min. After 48 h of dehydration in an environmental temperature of 20 degrees C the renal ADH excretion increased to about 8 mU/h, suggesting an 8-fold increase of ADH secretion over basic, water replete secretion of ADH. ADH secretion of the same high order was induced in hydrated animals by the infusion of angiotensin II together with hypertonic NaCl into the lateral cerebral ventricle.