Angiotensin, thirst, and sodium appetite.

Angiotensin (ANG) II is a powerful and phylogenetically widespread stimulus to thirst and sodium appetite. When it is injected directly into sensitive areas of the brain, it causes an immediate increase in water intake followed by a slower increase in NaCl intake. Drinking is vigorous, highly motivated, and rapidly completed. The amounts of water taken within 15 min or so of injection can exceed what the animal would spontaneously drink in the course of its normal activities over 24 h. The increase in NaCl intake is slower in onset, more persistent, and affected by experience. Increases in circulating ANG II have similar effects on drinking, although these may be partly obscured by accompanying rises in blood pressure. The circumventricular organs, median preoptic nucleus, and tissue surrounding the anteroventral third ventricle in the lamina terminalis (AV3V region) provide the neuroanatomic focus for thirst, sodium appetite, and cardiovascular control, making extensive connections with the hypothalamus, limbic system, and brain stem. The AV3V region is well provided with angiotensinergic nerve endings and angiotensin AT1 receptors, the receptor type responsible for acute responses to ANG II, and it responds vigorously to the dipsogenic action of ANG II. The nucleus tractus solitarius and other structures in the brain stem form part of a negative-feedback system for blood volume control, responding to baroreceptor and volume receptor information from the circulation and sending ascending noradrenergic and other projections to the AV3V region. The subfornical organ, organum vasculosum of the lamina terminalis and area postrema contain ANG II-sensitive receptors that allow circulating ANG II to interact with central nervous structures involved in hypovolemic thirst and sodium appetite and blood pressure control. Angiotensin peptides generated inside the blood-brain barrier may act as conventional neurotransmitters or, in view of the many instances of anatomic separation between sites of production and receptors, they may act as paracrine agents at a distance from their point of release. An attractive speculation is that some are responsible for long-term changes in neuronal organization, especially of sodium appetite. Anatomic mismatches between sites of production and receptors are less evident in limbic and brain stem structures responsible for body fluid homeostasis and blood pressure control. Limbic structures are rich in other neuroactive peptides, some of which have powerful effects on drinking, and they and many of the classical nonpeptide neurotransmitters may interact with ANG II to augment or inhibit drinking behavior. Because ANG II immunoreactivity and binding are so widely distributed in the central nervous system, brain ANG II is unlikely to have a role as circumscribed as that of circulating ANG II. Angiotensin peptides generated from brain precursors may also be involved in functions that have little immediate effect on body fluid homeostasis and blood pressure control, such as cell differentiation, regeneration and remodeling, or learning and memory. Analysis of the mechanisms of increased drinking caused by drugs and experimental procedures that activate the renal renin-angiotensin system, and clinical conditions in which renal renin secretion is increased, have provided evidence that endogenously released renal renin can generate enough circulating ANG II to stimulate drinking. But it is also certain that other mechanisms of thirst and sodium appetite still operate when the effects of circulating ANG II are blocked or absent, although it is not known whether this is also true for angiotensin peptides formed in the brain. Whether ANG II should be regarded primarily as a hormone released in hypovolemia helping to defend the blood volume, a neurotransmitter or paracrine agent with a privileged role in the neural pathways for thirst and sodium appetite of all kinds, a neural organizer especially in sodium appetit

Increased sodium appetite stimulates c-fos expression in the organum vasculosum of the lamina terminalis.

The relation between c-fos expression in the forebrain of Lister hooded rats and water and NaCl intakes was examined in response to systemic injection of angiotensin II, desoxycorticosterone, angiotensin II and desoxycorticosterone together, frusemide or low sodium diet, all treatments that induce a sodium appetite. Angiotensin II (1 mg/kg subcutaneously) caused significant increases in the 1-h intakes of water and 1.8% NaCl compared to controls, the effect on water intake being the greater. There was a similar increase in NaCl intake after four days' treatment with desoxycorticosterone (20 mg pellet subcutaneously) but water intake was not increased. The NaCl intake of rats given angiotensin II following desoxycorticosterone treatment was approximately the sum of the intakes after angiotensin II or desoxycorticosterone alone, but the water intake was slightly less than after angiotensin II alone. Frusemide pretreatment (4 mg/kg subcutaneously) caused an NaCl intake similar to that following angiotensin II and desoxycorticosterone but water intake was little affected. Low dietary sodium also increased salt appetite, as expected. These treatments were repeated in rats that were not allowed to drink NaCl, after which the brains were processed for c-fos immunocytochemistry. This showed intense staining of the subfornical organ, median preoptic nucleus, organum vasculosum of the laminal terminalis, paraventricular nucleus and supraoptic nucleus after subcutanous angiotensin II. Animals given angiotensin II following desoxycorticosterone pretreatment showed patterns of c-fos expression that did not differ from those of angiotensin II alone. Treatment with desoxycorticosterone alone produced intense staining in the organum vasculosum of the laminal terminalis and some staining in the median preoptic nucleus. Frusemide gave a similar pattern of staining to desoxycorticosterone, stimulating c-fos expression in the same regions but to a lesser extent. A low salt diet resulted in increased c-fos expression only in the organum vasculosum of the laminal terminalis. Therefore, five different treatments that induced increased sodium appetite evoked distinct patterns of c-fos expression in the anteroventral region of the third ventricle of the rat forebrain. Since the common feature was induction of c-fos in the organum vasculosum of the laminal terminalis, these results suggest a key role for this structure in the development of increased sodium appetite.

Increased sodium appetite and thirst in rat induced by the ingredients of liquorice, glycyrrhizic acid and glycyrrhetinic acid.

In the rat, blocking 11 beta-OHSD with the active ingredient of liquorice, glycyrrhizic acid (GZA) or its hydrolytic product, 18 beta-glycyrrhetinic acid (GTA), caused potassium loss, increased water intake and a primary increase in salt appetite that was specific for sodium and not secondary to sodium loss. Intracerebroventricular injection of angiotensin II enhanced the sodium appetite but carbachol did not. The stimulating effects of GZA or GTA on intakes of water and NaCl resembled those caused by the administration of excessive amounts of mineralocorticoid. The results suggest that GZA- or GTA-induced drinking behaviour is mediated by circulating glucocorticoids. After liquorice blockade of 11 beta-OHSD, the peripheral and central mineralocorticoid receptors are no longer protected from glucocorticoid action.

The association of thirst, sodium appetite and vasopressin release with c-fos expression in the forebrain of the rat after intracerebroventricular injection of angiotensin II, angiotensin-(1-7) or carbachol.

The effect intracerebroventricular injections of angiotensin II (0.1 nm), angiotensin-(1-7) (1 or 100 nm) and carbachol (500 ng) on c-fos expression was examined in the forebrain of Lister hooded rats. Intense staining of the c-Fos protein was found in the median preoptic nucleus, organum vasculosum of the lamina terminalis, subfornical organ, paraventricular nucleus and supraoptic nucleus after angiotensin II and carbachol Angiotensin II caused significantly more c-fos expression in the ventral median preoptic nucleus and organum vasculosum of the lamina terminalis than carbachol, whereas in the paraventricular and supraoptic nuclei this was reversed, with carbachol having a greater effect on c-fos expression in these areas. Angiotensin-(1-7), however, only induced c-Fos protein in the organum vasculosum of the lamina terminalis and median preoptic nucleus with the number and the intensity of staining of the nuclei significantly less in both areas than after angiotensin II or carbachol. Separate groups of Lister rats were given i.c.v. injections of the same substances at the same doses, but excluding the lower dose of angiotensin-(1-7), and the intakes of water and 1.8% NaCl over 60 min were measured. Angiotensin II stimulated intakes of both water and NaCl. The effect on water intake was almost immediate (<1 min), whereas NaCl intake did not usually start until at least 5 min after injection. Over 60 min, water (12.4 +/- 1.0 ml) and NaCl (4.2 +/- 0.9 ml) intakes were significantly greater than water (1.1 +/- 0.2 ml) and NaCl (0.6 +/- 0.5 ml) intakes of the controls. Carbachol caused less drinking than angiotensin II, the water intake over 60 min being significantly less (4.8 +/- 0.7 ml) and the latency of response greater (>5 min). Carbachol, unlike angiotensin II, had little effect on NaCl intake (0.7 +/- 0.4 ml). Angiotensin-(1-7) had no effect on water (1.1 +/- 0.3 ml) or NaCl (0.3 +/- 0.3 ml) intakes. The plasma levels of vasopressin were measured after i.c.v. injection of the same three substances in the same doses, again excluding the lower dose of angiotensin-(1-7), in further groups of rats. Angiotensin II and carbachol caused an approximate five-fold increase in plasma vasopressin levels compared to cerebrospinal fluid-injected rats, but angiotensin-(1-7) had no effect on vasopressin release. Therefore, three compounds with widely differing effects on thirst, sodium appetite and vasopressin release induce distinctive patterns of c-fos protein expression in the forebrain. By combining experimental approaches in this way it is possible to determine areas of the brain which are involved in certain behavioural and endocrine responses.

The effects of centrally administered porcine relaxin on drinking behaviour in male and female rats.

Of the reproductive hormones it has been suggested that relaxin may play an important role in the increased sodium appetite of pregnancy. ICV injection of porcine relaxin caused water-replete male and female Wistar rats with access to water and 0.9% or 2.7% NaCl to drink on average about 3 to 8 ml of water within 1 h of injection. By 24 h the cumulative intake of water was no different from the control intake. The amounts of water drunk were similar after doses of 50, 100, 250 and 500 ng of relaxin. A dose of 5 ng was ineffective. Male rats generally drank more water than female rats after ICV injection of angiotensin or relaxin. Male SH rats which drink more water than male WKY rats in response to ICV angiotensin also drank more after ICV relaxin. Intakes of 0.9% or 2.7% NaCl were unaffected for up to 24 h after injection of relaxin, whereas angiotensin-injected rats showed a significant increase in 0.9% NaCl 1 h after injection though this difference was no longer evident in the 24 h cumulative intake. Relaxin did not cause any increase in NaCl intake in SH rats. Insulin, which is similar in structure and molecular weight to relaxin, was without effect on drinking when doses comparable to dipsogenically effective doses of relaxin were injected ICV. In male Wistar rats treated with DOCA for 5-15 days, relaxin retained its weak stimulatory action on water intake but did not affect NaCl intake despite the increased baseline NaCl intake during DOCA. These results indicate that relaxin is a dipsogen in the rat but that it seems to have little short-term effect on sodium appetite.

The effect of the putative AT2 agonist, p-aminophenylalanine6 angiotensin II, on thirst and sodium appetite in rats.

Intracerebroventricular injection of the putative AT2 agonist, p-aminophenylalanine6 angiotensin II (p-NH2Phe6-Ang II), caused dose-dependent increases in intakes of water and NaCl similar to those produced by angiotensin II but requiring more than one thousand times the dose. Very large doses of another AT2 agonist, angiotensin(1-7) heptapeptide (Ang(1-7)), had no effect on intakes of water and NaCl up to 24 h after injection, nor did Ang(1-7) affect angiotensin II-induced drinking when the two peptides were given together. The AT1 antagonist, losartan, but not the AT2 antagonist, CGP 42112B, inhibited p-NH2Phe6-Ang II- and angiotensin II-induced drinking, suggesting that p-NH2Phe6-Ang II, like angiotensin II, acts on AT1 but not AT2 receptors. However, large doses of the AT2 antagonist, PD 123319, inhibited drinking in response to both dipsogens. Since p-NH2Phe6-Ang II- and angiotensin II-induced drinking were unaffected by CGP 42112B, this could mean that there are different AT2 receptor subtypes of which only the PD 123319-sensitive one is involved in drinking. But because of the very large doses of PD 123319 used it is also likely that there was loss of receptor specificity resulting in cross-reaction of PD 123319 with AT1 receptors. The results do not favour involvement of AT2 receptors in angiotensin-induced thirst and sodium appetite in the short term.

Intracerebroventricular angiotensin II-induced thirst and sodium appetite in rat are blocked by the AT1 receptor antagonist, Losartan (DuP 753), but not by the AT2 antagonist, CGP 42112B.

In the rat, intakes of water and 1.8% NaCl induced by I.C.V. angiotensin II were inhibited by prior I.C.V. injection of the angiotensin subtype 1 receptor antagonist, Losartan, but not by the subtype 2 receptor antagonist, CGP 42112B. Drinking induced by I.C.V. carbachol was unaffected by either antagonist.

Effects of soyabean and ascorbic acid on experimental carcinogenesis.

1. Ultrastructural changes in liver tissue of mice fed nitrosamine precursors, dibutylamine and nitrite, were observed. 2. The protective effect of soyabean in a diet containing nitrosamine precursors was demonstrated. 3. Liver tissue was examined to investigate the anticarcinogenicity of ascorbic acid. 4. The significance of soyabean and ascrobic acid in counteracting the potential hazards due to nitrosamine precursors is discussed.

Thirst in Brattleboro rats.

Thirst mechanisms in Brattleboro rats are activated because of a deficiency in circulating vasopressin. Plasma osmolality, renin, and angiotensin II (ANG II) are increased. We measured the responsiveness of Brattleboro rats and appropriate control strains to cellular and extracellular thirst stimuli taking the spontaneous base-line water intake into account. Brattleboro rats drank more in response to intraperitoneal hypertonic NaCl than controls, but when their fluid losses were prevented by nephrectomy they did not overdrink. Despite low urinary concentration, Brattleboro rats excreted the sodium load at least as rapidly as the controls. Brattleboro rats drank after intracranial injection of renin, renin substrate, and ANG I and II. The dose-response curves were similar to controls, although the Nottingham Long-Evans control strain drank significantly less in response to some doses of the peptides. Intracranial captopril inhibited renin- and ANG I-induced but not ANG II-induced drinking. Isoproterenol reduced spontaneous drinking of Brattleboro rats but increased drinking in controls. However, when urinary losses were prevented by ureteric ligation, isoproterenol caused markedly greater water intake in Brattleboro rats than in controls. Subcutaneous captopril in moderate, thirst-enhancing doses also caused a larger increase in water intake in Brattleboro rats than in controls. Therefore the renin-angiotensin system of Brattleboro rats is more responsive to renin-dependent thirst challenges than that of normal controls.

The effect of captopril on sodium appetite in adrenalectomized and deoxycorticosterone-treated rats.

Captopril caused a renin-dependent increase in water intake in rats with bilateral ureteric ligation. But despite the fluid retention and fall in osmolality caused by the increased water intake, rats with ureteric ligation did not drink the 2.7% NaCl also offered to them. In rats with a pre-existing increase in sodium appetite caused by adrenalectomy, low dosage of captopril augmented intake of both water and 2.7% NaCl whereas high dosage inhibited intake of both fluids after an initial increase in water intake. In contrast, rats with a pre-existing increase in sodium appetite caused by daily injections of deoxycorticosterone showed no changes in intake of water or 2.7% NaCl after either low or high dosage of captopril, though they drank both fluids after intracranial injection of angiotensin II. Increases in water and 2.7% NaCl intake caused by low dosage of captopril in adrenalectomized rats were not secondary to increased urinary fluid and electrolyte losses. Decreases in intake after high dosage were not explained by the rats being too weak to drink. Low and high dosage of captopril caused increases in plasma renin concentration in adrenalectomized rats, but in contrast renin remained undetectable in the plasma of deoxycorticosterone-treated rats after the highest dosage of captopril. Whether or not captopril affected a pre-existing sodium appetite depended on whether or not it increased plasma renin. Since it affected the pre-existing sodium appetite and plasma renin in the adrenalectomized rat but neither of these in the deoxycorticosterone-treated rat, it is likely that the appetite was renin-dependent in the former but not in the latter. Because captopril only affected thirst in the rat with ligated ureters whereas it affected both sodium appetite and thirst in the adrenalectomized rat, increases in renal renin secretion alone may not be enough to stimulate sodium appetite. The additional stimulus provided by adrenalectomy, or the absence of some inhibitory factor that may be present after ureteric ligation, is also needed.

Effects of angiotensin or carbachol on sodium intake and excretion in adrenalectomized or deoxycorticosterone-treated rats.

In adrenalectomized, deoxycorticosterone-treated and normal rats, injection of angiotensin II through a cannula implanted in the preoptic region caused increased intakes of hypertonic NaCl and water when both fluids were available, whereas injection of carbachol through the same cannula only caused increased water intake. Carbachol depressed NaCl intake of adrenalectomized rats that were allowed access to hypertonic NaCl after being deprived of it for 24 h. Angiotensin-stimulated rats were more likely to go into positive sodium balance than controls, whereas carbachol-stimulated animals were more likely to go into negative balance. After angiotensin, adrenalectomized or deoxycorticosterone-treated rats drank a larger proportion of their total fluid intake as hypertonic NaCl than did normal rats. Angiotensin caused significant increases in sodium excretion in normal, isotonic saline-loaded and deoxycorticosterone-treated rats, but not in adrenalectomized rats, although angiotensin caused increased intakes of NaCl in all groups. On the other hand, carbachol caused a significant increase in sodium excretion at 1 h in all groups despite the absence of an increase in NaCl intake. After angiotensin, only normal rats showed a significant kaliuresis at 1 h, whereas all carbachol-injected rats showed increased potassium excretion. Therefore, angiotensin is a primary stimulus to increased sodium appetite, normally acting in conjunction with other stimuli which enhance its effect, whereas carbachol is a central inhibitor of sodium appetite.

Structural effects of external media on isolated myelinated axons.

Myelinated axons were isolated from the sciatic nerve of Xenopus laevis by desheathing and teasing, and were mounted for short-term light microscope observation in different media. Fibres mounted in a conventional physiological saline showed a tendency to gross structural changes, including collapse of axons and separation of the axon from the myelin sheath. With isotonic media based on 120mM potassium aspartate, or 120mM potassium glutamate, structural stability and axoplasmic function (assessed by persistence of particle transport) were well maintained. The speeds of retrograde particle transport in fibres immersed in potassium aspartate or potassium glutamate medium ranged from 0.05 to 3.15 micron/sec with mean speeds of 1.05 and 1.2 micron/sec. Transport persisted for up to 3 hr. Isotonic media based on amino acids but lacking K+ or Na+ were unsatisfactory short-term culture media and destabilized myelin structure. Inclusion of 60 mM KCl in the medium reduced structural disruption and allowed particle transport to persist.

Involvement of the renin-angiotensin system in captopril-induced sodium appetite in the rat.

The angiotensin converting enzyme inhibitor, captopril, given to rats in their drinking water (about 40 mg/day) for 6 days caused an increase in intake of hypertonic NaCl solution which began 1-2 days after the captopril was started and reached a plateau after 4-5 days. Twice-daily subcutaneous injections of captopril (15 mg per injection) elicited a sodium appetite similar in pattern to that seen with oral administration. The rats remained in sodium and fluid balance during oral captopril treatment and the haematocrit did not alter. Captopril infused directly into the ventricles (12 micrograms/h), or captopril reaching the brain from the periphery across a leaky blood-brain barrier, suppressed the sodium appetite which normally follows oral captopril. Continuous intravenous infusion of captopril at rates high enough to block angiotensin converting enzyme in the brain (25, 50 or 500 mg/day) did not cause sodium appetite. As soon as the rate was reduced to a low value (5 mg/day), NaCl intake increased. In conclusion, moderate levels of circulating captopril which do not cross the blood-brain barrier in sufficient amounts to block cerebral angiotensin converting enzyme, result in an increase in circulating angiotensin I which stimulates sodium appetite when it is converted to angiotension II in the brain.

Increased sodium appetite and polydipsia induced by partial aortic occlusion in the rat.

Partly occluding the abdominal aorta between the renal arteries caused the rat to drink steadily increasing amounts of 2.7% NaCl when this solution and water were available. The increase in NaCl intake preceded the increase in water intake that also occurred after aortic occlusion, and intakes of both fluids were reaching maximal values 1-2 weeks after operation. The amounts of fluid drunk during the day increased greatly. This change in the pattern of drinking, together with the rise in fluid intake and the drop in food intake meant that drinking was less associated with feeding than it is in the normal rat. The rats went into fluid and electrolyte deficit within 24 h of partial aortic occlusion and remained in deficit for about a week (the duration of the balance experiment) despite increasing intakes of NaCl and water. Renal function was unimpaired during the first 2 weeks, and the abnormal signs were mainly and rapidly reversed by removal of the ischaemic kidney or administration of the angiotensin converting enzyme inhibitor, captopril. Therefore polydipsia and increased sodium appetite in the first 2 weeks after aortic occlusion were likely to have been caused by fluid deficit, with increased renin secretion from the ischaemic kidney contributing to both behaviours. Arterial blood pressure rose immediately after aortic occlusion, before the onset of increased drinking. Up to 3 weeks after operation the incidence and severity of the hypertension did not appear to depend on the spontaneous changes in intake of water or hypertonic NaCl.

The renin-angiotensin system and sodium appetite.

Intracranial renin is a potent stimulus to sodium appetite and thirst, the effects being mediated by local generation of angiotensin II. Intakes are persistent and lead to fluid retention during the first 24 h (Avrith and Fitzsimons, 1983). Increased circulating renin after captopril treatment in adrenalectomized rats (Elfont and Fitzsimons, 1981), or in renal hypertension following partial inter-renal aortic ligation (Costales et al., 1982), also leads to increased intakes of 2.7% NaCl and water. Fluid intakes after aortic ligation were independent of the severity of hypertension produced by this procedure. In both the examples given, additional stimulation resulting from the hypovolaemia itself is required for the full expression of increased sodium appetite, but in both cases angiotensin makes a significant contribution to sodium appetite as well as thirst. Therefore, as has been shown for thirst, angiotensin is one of a number of factors that act together to cause increased sodium appetite in hypovolaemia.

Renin dependence of captopril-induced drinking after ureteric ligation in the rat.

In experiments lasting 8 h, low (0.5 mg kg-1) or medium (5 mg kg-1) subcutaneous doses of the angiotensin-converting enzyme inhibitor captopril were mildly dipsogenic in sham-operated rats, much more so in rats subjected to bilateral ureteric ligation and not at all in bilaterally nephrectomized rats. Rats with ligated ureters drank enough water to gain weight during the experiments. All other groups lost weight. The enhanced responsiveness of rats with ligated ureters, despite fluid retention, shows that captopril-induced drinking was not secondary to increased renal fluid loss. Ureteric ligation alone which caused some increase in renin secretion was mildly dipsogenic compared with sham operation. Captopril caused further increases in plasma renin concentration and more drinking suggesting that the captopril response is renin-dependent. The failure of the nephrectomized rat to drink after captopril also shows that the response is renin-dependent. The highest dose (50 mg kg-1) of captopril did not at first stimulate drinking, though water intake increased later. Slowness to drink was not the result of general depression of behaviour since drinking in response to subcutaneous hypertonic NaCl or intracranial angiotensin II was not inhibited by the highest dose. Slowness to drink after the highest dose was attributable to blockade of converting enzyme centrally as well as peripherally. This meant that the increased circulating angiotensin I resulting from peripheral blockade of converting enzyme was only slowly converted to angiotensin II in the brain. When cerebral conversion of angiotensin I was prevented by a single intracranial injection of 25 micrograms captopril, drinking in response to the lower doses of captopril was also inhibited in normal rats and in rats with ligated ureters. The same intracranial dose of captopril also inhibited drinking in response to intracranial injections of renin or angiotensin I, but not angiotensin II. The time course of inhibition of renin-induced drinking was similar to that of inhibition of subcutaneous captopril-induced drinking. In conclusion, subcutaneous captopril causes increased water intake through activation of the renal renin-angiotensin system, an effect that is enhanced when the system has already been partly activated by ureteric ligation. Increased circulating angiotensin I resulting from blockade of peripheral converting enzyme must be converted to angiotensin II in the brain in order to stimulate drinking. Drinking is not the consequence of increased fluid loss.

The effects of corticosterone, cold exposure and overfeeding with sucrose on brown adipose tissue of obese Zucker rats (fa/fa).

GDP binding to brown-adipose-tissue mitochondria was decreased in obese Zucker rats. Adrenalectomy restored both GDP binding and serum tri-iodothyronine of obese rats to values observed in lean rats. The effects of adrenalectomy on GDP binding and serum tri-iodothyronine were reversed by corticosterone. Decreasing food intake had no effect on brown-adipose-tissue GDP binding in obese rats. Young (5-week-old) obese rats showed a normal increase in brown-adipose-tissue mitochondrial GDP binding after housing at 4 degrees C for 7 days, but this response was attenuated in 10-week-old obese rats. Overfeeding with sucrose increased brown-adipose-tissue thermogenesis in lean, but not in obese, rats. After adrenalectomy, overfeeding with sucrose enhanced brown-adipose-tissue mitochondrial GDP binding in obese rats.