Effects of the urinary natriuretic fraction on smooth muscle and its further purification
The experiments in this dissertation were undertaken to purify a humoral natriuretic substance from human urine and to investigate its possible role in hypertension by examining its effect on smooth muscle at various stages of purification. Activity of the natriuretic substance was monitored by its ability to cause sodium excretion in conscious rats and by its effect on Na-KATPase which mediates sodium transport in epithelial tissues including renal tubules. The natriuretic substance was purified from human urine by successive purifications on sephadex G-25 anf G-10 gels. Further purification was attempted with cation exchange resin AG-50W x8. The NF (natriuretic fraction) of sephadex G-25 contracted smooth muscle and the contracting activity of the NF positively correlated with its naturiuretic activity. Also both the contracting and the natriuretic activities were excreted in higher concentrations in hypertensive subjects. These findings, taken in isolation, support the hypotheses of Haddy et al (1976) and Balustein (1977). However, further purification of the NF on sephadex G-10 revealed the presence of two smooth muscle contracting substances and one natriuretic substance in the NF of sephadex G-25. The two contracting substances were not natriuretic and they stimulated Na-KATPase. The natriuretic substance did not contract smooth muscle although it inhibited Na-KATPase. Thus the natriuretic substance of human urine (extracted by sephadex G-10) is most unlikely to be the sodium transport inhibitor postulated (Haddy et al , 1976; Blaustein, 1977) to have a role in the genesis of hypertension. It is known that a subgroup of hypertensive subjects has raised plasma catecholamine levels. The contracting activity of sephadex G-25 extracted NF was found to be excreted in higher concentrations in hypertensive subjects. Further purification of NF on sephadex G-10 showed that the contracting activity of NF was due to two contracting substances, one of which was a catecholamine and the other could be a derivative of catecholamines. Further studies and purification would be necessary to establish the identity of these catecholamines and to ascertain which or whether both of these two catecholamines are excreted in higher concentrations in hypertension and what role they could play in the pathogenesis of hypertension. Similarly, the natriuretic substance was also excreted in higher concentration in hypertension. Additional studies would be necessary to delineate the possible role of the natriuretic substance in the impaired ability of the kidney to handle sodium in hypertension. The NF of sephadex G-25 contained both stimulators and inhibitors of Na-KATPase. With continued use of the same sephadex G-25 column, more Na-KATPase inhibitory and less stimulatory activity tend to elute. Purification of the NF of sephadex G-25 on sephadex G-10 showed the presence of multiple Na-KATPase stimulators in the NF. An intense stimulator of Na-KATPase was found in both sephadex G-25 and G-10 stages of purification. Purification on sephadex G-10 revealed that this intense stimulator of Na-KATPase caused significant antinatriuresis. Purification on sephadex G-10 also showed that there were two Na-KATPase inhibitors in the NF of sephadex G-25 but only the pre-salt Na-KATPase inhibitor caused natriuresis. Thus the antinatriuretic intense stimulator of Na-KATPase with the natriuretic inhibitor of Na-KATPase could form the physiological regulatory system for Na-KATPase. Sephadex G-10 was a useful purification medium as shown by 8-fold increase in the specific activity of the natriuretic substance. However, cation exchange resin was not a useful medium for further purification as evidenced by the lack of increase of the specific activity of the natriuretic substance and by the elution pattern of the natriuretic substance. The natriuretic substance is likely to have a molecular weight of less than 700 daltons since it eluted in pre-salt fraction of sephadex G-10.