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SUMMARY
N-Ethylmaleimide (NEM) prevents the hydro-osmotic response to vasopressin. It has no significant effects on osmotic water movement in the absence of the hormone. The hydro-osmotic effects of cyclic AMP and hyperosmotic mannitol solutions are also reduced by NEM.
After exposure to vasopressin, cyclic AMP, or hyperosmotic mannitol, the osmotic permeability declines to the preincubation level but after exposure to NEM this was prevented. This effect was seen after exposure to 10−4 and 10−5 m-NEM, i.e. concentrations that do not alter the onset of the response.
The increased osmotic permeability after treatment with NEM is not due to a non-specific effect caused by a 'fixation' of the tissue in a distorted condition (such as occurs during rapid water transfer) since it was seen even after treatment of the tissue under conditions when no osmotic water movement was occurring, that is with iso-osmotic Ringer solution on both sides.
The effect of NEM could not be mimicked by exposure of the bladder to 20 mm-Ca2+, 0·1 mm-Zn2+ or 1 mm-l-cysteine.
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SUMMARY
The effects of vasopressin analogues with selective antidiuretic activity have been tested on the isolated toad bladder.
Analogues of oxytocin and 8-Arg vasopressin lacking the terminal amino group had reduced effects on water (hydro-osmotic effect) and sodium (natriferic effect) transfer across the bladder. The natriferic effect of deamino-8-Arg vasopressin was reduced 3·5 times more than its hydro-osmotic effect.
Deamino-2-Phe, 8-Arg vasopressin had a reduced hydro-osmotic action compared with deamino-8-Arg vasopressin (6 times less) while the reduction of the natriferic effect was far more pronounced (68 times).
The selective antidiuretic, compared to pressor, potency of these analogues is discussed in relation to their selective hydro-osmotic compared to natriferic potency.
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SUMMARY
The short-circuit current (SCC, sodium transport) across the isolated toad urinary bladder was increased by the presence of 10−4 m-Mn2+ at the serosal but not at the mucosal surface. Magnesium did not have this effect. Zinc (10−4 m) decreased the SCC. Neither Mn2+ nor Zn2+ altered the SCC at a concentration of 10−5 m.
Oxytocin stimulated sodium transport across the bladder and this response was reduced by 10−4 but not by 10−5 m-Mn2+. Zinc (10−5 m) nearly abolished the effect of oxytocin.
Normal osmotic permeability of the bladder to water was unaffected by 10−4 m-Mn2+ or Zn2+.
Oxytocin increased the osmotic permeability of the bladder to water and this response was strongly inhibited by 10−4 and 10−5 m-Mn2+ at the serosal but not at the mucosal surface. Magnesium did not have this effect. Zinc (10−4 to 10−6 m) similarly reduced this effect of oxytocin but at a concentration of 10−4 m also acted at the mucosal surface.
The antagonistic effect of Mn2+ on the action of oxytocin on osmotic permeability was not reversible and was non-competitive. Zn2+ was also found to be a non-competitive antagonist of oxytocin but the antagonism was reversible.
The increase in osmotic permeability of the bladder to water by the addition of cyclic AMP was unaffected by Mn2+. In contrast Zn2+ reduced the effects of cyclic AMP on both osmotic permeability and SCC. It is suggested that Mn2+ uncouples processes linking the interaction of oxytocin with the formation of cyclic AMP in the bladder while Zn2+ acts at a stage subsequent to the formation of the nucleotide, possibly on the effector mechanisms themselves.
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SUMMARY
The electrical potential difference and short-circuit current (scc, reflecting active transmural sodium transport) across the toad urinary bladder in vitro was unaffected by the presence of hypo-osmotic solutions bathing the mucosal (urinary) surface, providing that the transmural flow of water was small.
Vasopressin increased the scc across the toad bladder (the natriferic response), but this stimulation was considerably reduced in the presence of a hypo-osmotic solution on the mucosal side, conditions under which water transfer across the membrane was also increased.
This inhibition of the natriferic response did not depend on the direction of the water movement, for if the osmotic gradient was the opposite way to that which normally occurs, the response to vasopressin was still reduced.
The natriferic response to cyclic AMP was also inhibited in the presence of an osmotic gradient. Aldosterone increased the scc and Na+ transport across the toad bladder but this response was not changed when an osmotic gradient was present.
The physiological implications of these observations and the possible mechanisms involved are discussed.
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SUMMARY
Ethacrynic acid inhibited short-circuit current (SCC, Na transport) across the toad bladder. Some structural analogues had similar effects and their potency varied with their ability to combine with SH groups. The Na-K ATPase inhibitor ouabain was more effective than ethacrynic acid and its action and that of ethacrynic acid was additive.
The natriferic and hydro-osmotic effects of vasopressin were inhibited by ethacrynic acid (10−5 and 10−4 m); the antagonism was competitive at 10− and 10−4 m (but not at 10−3 m). Certain analogues of ethacrynic acid also exhibited this effect but it was not clearly related to their SH-combining ability.
The natriferic and hydro-osmotic effects of caffeine and cyclic AMP were not changed by 10−4 m-ethacrynic acid.
Ethacrynic acid (10−4 m), which does not affect basal sodium transport in bladders pretreated for studying the action of aldosterone, prevented the initiation of the increase in sodium transport by aldosterone but was without effect when added to a toad bladder already under the influence of aldosterone. This suggests that ethacrynic acid interferes with metabolic processes underlying the action of aldosterone. Dihydroethacrynic acid which has no SH-binding activity was without effect.
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SUMMARY
Blood plasma of dehydrated toads (Bufo marinus) and bullfrogs (Rana catesbeiana) increased water transfer (hydro-osmotic response) across the isolated toad urinary bladder. Sodium transport (in terms of the shortcircuit current) was also increased.
Immersion of Bufo marinus, Rana catesbeiana and Xenopus laevis (but not the neotenous newt Necturus maculosus) for 2 hr. in a 2% NaCl solution also increased the hydro-osmotic activity of plasma. Ether and haemorrhage also had this effect on plasma from Rana and Bufo.
Hydro-osmotic activity of plasma from dehydrated bullfrogs was destroyed by incubation with trypsin or tyrosinase, evidence which combined with the pharmacological effects of the plasma indicate that the hydroosmotic activity in the plasma is due to arginine vasotocin. The concentrations of this hormone were comparable with levels of antidiuretic activity observed in mammalian plasma.
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SUMMARY
Analogues of oxytocin contract the rat uterus in vitro more strongly in the presence of 0·5 mm-Mg2+ than in the absence of the ion. This effect can be referred to an increased 'affinity' of the hormone for its receptors. Neither Ca2+ (1 mm), Sr2+ (0·5 mm) nor lower concentrations of Mg2+ (0·1 mm) have this effect. Manganese mm) was more effective than Mg2+ (0·5 mm); the former potentiated the action of 3-valine oxytocin 9·9 times; that of 8-arginine oxytocin 3·3 times and that of 8-isoleucine oxytocin 1·3 times while 0·5 mm-Mg2+ potentiated the same analogues 5·7, 1·9 and 1·1 times, respectively. The results are discussed in relation to the mechanism of action of oxytocin.
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SUMMARY
1. The effect of oxytocin on both the rat uterus and frog bladder is abolished by solutions of 1 mm N-ethylmaleimide (NEM).
2. NEM, however, has a variety of effects on these tissues. It inhibits sodium transport across the bladder, it prevents relaxation of the uterus and the return of water transfer to normal after it has been affected by oxytocin, and it inhibits contraction of the uterus by acetylcholine.
3. Reduced and oxidized glutathione (GSH and GSSG) inhibit the action of oxytocin on the uterus reversably and this inhibition is competitive.
4. GSH and GSSG also inhibited the increase in water transfer by oxytocin across the frog bladder, GSSG was more potent. Sodium transport across the bladder was transiently reduced by GSH and decreases by about 50% in the presence of GSSG.
5. The results are discussed in relation to the possible mechanism of interaction of neurohypophysial hormones with their receptors. It is concluded that both S-S and SH groups are present at the receptor sites in both the rat uterus and frog bladder.
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In mammals, neurohypophysial hormones play a physiological role in the regulation of the metabolism of water and influence the contractility of certain smooth muscles. They may also produce hyperglycaemia (see Waring & Landgrebe, 1950; Mirsky, 1963; Cash & Kaplan, 1964). It is not known whether this response is of physiological significance. A hyperglycaemic effect has also recently been shown when the homologous neurohypophysial hormone, arginine vasotocin, is injected into lampreys (Cyclostomata) and toads (Amphibia) (Bentley & Follett, 1965; Bentley, 1965) and when oxytocin is given i.v. to chickens (Kook, Cho & Yun, 1964). The neurohypophysis of the chicken (Gallus domesticus) contains oxytocin, and vasotocin is also present but at five times greater concentration (Munsick, Sawyer & van Dyke, 1960; Heller & Pickering, 1961). Vasotocin is much more active than oxytocin in producing antidiuresis and contraction of the oviduct in this species (Munsick et al. 1960). The present study confirms the
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Arginine-vasotocin (8-arginine-oxytocin, AVT) has been found in the neuro-hypophyses of nearly all vertebrate classes that are lower in the phylogenetic scale than mammals. This hormone has a number of pharmacological properties in common with oxytocin (such as an ability to contract the rat uterus) as well as with vasopressin (ability to increase the blood pressure of anaesthetized rats). AVT has, however, a greater ability than either of these hormones to increase sodium transport (natriferic activity) across the isolated urinary bladder and skin of anurans (see Heller, Pickering, Maetz & Morel, 1961). The present work describes a method for the estimation of AVT in biological materials using the natriferic response of the frog bladder.
The procedure for setting up the preparation of the isolated anuran bladder has been described in detail previously (Bentley, 1958). Frog (Rana esculenta) bladders were tied to the end of a piece of glass tubing, filled with
