M. McBurnie

The relaxin gene-knockout mouse : A model of progressive fibrosis

Abstract: Relaxin is well known for its actions on collagen remodeling. To improve our understanding of the physiologic role(s) of relaxin, the relaxin gene‐knockout (RLX‐KO) mouse was established by our group and subsequently phenotyped. Pregnant RLX‐KO mice underwent inadequate development of the pubic symphysis as well as the mammary glands and nipples compared to wild‐type mice, thus preventing lactation. Later studies showed that these deficiencies were associated with increased collagen, primarily in the nipple and vagina. Analysis of male RLX‐KO mice also demonstrated inadequate reproductive tract development. The testis, epididymis, and prostate of RLX‐KO mice showed delayed tissue maturation and growth associated with increased collagen deposition. In nonreproductive tissues, an age‐related increase in interstitial collagen (fibrosis) was also detected in the lung, heart, and kidneys of RLX‐KO mice and was associated with organ dysfunction. From 6‐9 months of age and onwards, all organs of RLX‐KO mice, particularly male mice, underwent progressive increases in tissue weight and collagen content (all P <.05) compared with wild‐type animals. The increased fibrosis contributed to bronchiole epithelium thickening and alveolar congestion (lung), atrial hypertrophy and increased ventricular chamber stiffness (heart) in addition to glomerulosclerosis (kidney). Treatment of RLX‐KO mice with recombinant human relaxin in early and developed stages of fibrosis caused the reversal of collagen deposition in the lung, heart, and kidneys. Together, these findings suggest that relaxin is a naturally occurring inhibitor of collagen deposition during normal development, aging, and pregnancy and can be used to prevent the progression of fibrosis.

Thirst induced by increasing brain sodium concentration is mediated by brain angiotensin

Thirst, the longing or compelling desire to drink, arises physiologically by two main mechanisms-extracellular and cellular dehydration. The hormone angiotensin II has been implicated in the former but not in the latter brain mechanism. To test this apparent difference, experiments in 5 mammalian species examined the effect of intracerebroventricular infusion of losartan, an angiotensin II type I receptor antagonist, on the third induced by intracerebroventricular infusion of an artificial cerebrospinal fluid made hypertonic by the inclusion of 500 mM NaCl. The losartan infusion reduced the water intake due to increased brain sodium concentration in all 5 species, cattle, sheep, rabbits, rats and mice. Thus, the thirst evoked by cellular dehydration, as well as the thirst evoked by extracellular dehydration, may be mediated by angiotensin II.

Neural Mechanisms Subserving Central Angiotensinergic Influences on Plasma Renin in Sheep

Abstract—The mechanisms and brain regions subserving the suppression of plasma renin concentration caused by intracerebroventricular (ICV) infusion of angiotensin II were studied in sodium-depleted sheep. Infusion of angiotensin II (3 &mgr;g/h for 1 hour) into the lateral ventricle reduced plasma renin from 4.3±0.4 to 1.6±0.2 pmol angiotensin I/mL per hour at 1 hour after the commencement of infusion. This change persisted for at least another 90 minutes and was blocked by concomitant ICV infusion of the AT1 antagonist losartan (1 mg/h). Arterial pressure did not change, but plasma vasopressin secretion was increased. ICV infusion of losartan (1 mg/h) significantly increased plasma renin in sodium-depleted sheep. The reduction of plasma renin concentration in response to either ICV angiotensin II or hypertonic NaCl (0.75 mol/L at 1 mL/h) and the increase in response to ICV losartan was prevented in sheep in which the lamina terminalis of the brain had been ablated. Lesions in the median eminence (MEL), which blocked the increased plasma vasopressin levels, did not prevent suppression of plasma renin in response to ICV angiotensin II. However, bilateral renal denervation largely blocked this inhibition of plasma renin concentration but not the increased plasma renin resulting from ICV infusion of losartan in sodium-depleted sheep. The results show that AT1 receptors, probably located in the lamina terminalis, mediate a central inhibitory influence of angiotensin II on renin secretion. This inhibition of renin release is probably due to a reduction in activity of renal sympathetic nerves innervating the juxtaglomerular apparatus of the kidney.

Effect of adrenocorticotrophic hormone on sodium appetite in mice

A main vector of the effects of stress is secretion of corticotrophin releasing factor (CRF), adrenocorticotrophin (ACTH), and adrenal steroids. Systemic administration of ACTH (2.8 microgram/day sc) for 7 days in BALB/c mice caused a very large increase of voluntary intake of 0.3 M NaCl equivalent to turnover of total body sodium content each day. Intracerebroventricular infusion of ACTH (20 ng/day) had no effect. Intracerebroventricular infusion of ovine CRF (10 ng/h for 7 days) caused an increase of sodium intake. The large sodium appetite-stimulating effect of systemic ACTH was not influenced by concurrent systemic infusion of captopril (2 mg/day). Induction of stress by immobilization of mice on a running wheel caused an increase in Na appetite associated with a 50% decrease of thymus weight, indicative of corticosteroid effects. The present data suggest that stress and the hormone cascade initiated by stress evoke a large sodium appetite in mice, which may be an important survival mechanism in environmental conditions causing stress.A main vector of the effects of stress is secretion of corticotrophin releasing factor (CRF), adrenocorticotrophin (ACTH), and adrenal steroids. Systemic administration of ACTH (2.8 μg/day sc) for 7 days in BALB/c mice caused a very large increase of voluntary intake of 0.3 M NaCl equivalent to turnover of total body sodium content each day. Intracerebroventricular infusion of ACTH (20 ng/day) had no effect. Intracerebroventricular infusion of ovine CRF (10 ng/h for 7 days) caused an increase of sodium intake. The large sodium appetite-stimulating effect of systemic ACTH was not influenced by concurrent systemic infusion of captopril (2 mg/day). Induction of stress by immobilization of mice on a running wheel caused an increase in Na appetite associated with a 50% decrease of thymus weight, indicative of corticosteroid effects. The present data suggest that stress and the hormone cascade initiated by stress evoke a large sodium appetite in mice, which may be an important survival mechanism in environmental conditions causing stress.

The effect of urocortin on ingestive behaviours and brain Fos immunoreactivity in mice

The influence of urocortin (UCN) on ingestive behaviours and brain neural activity, as measured immunohistochemically by the presence of Fos protein, was determined in mice. Rat UCN was administered by continuous intracerebroventricular (ICV) or subcutaneous (SC) infusion. ICV infusion of UCN (100 ng/h, 14 days) transiently reduced daily food and water intakes (days 1–4) but body weight was reduced from day 2 into the post‐infusion period. Sodium intake was reduced from day 3 to the end of infusion. SC infusion of UCN caused similar but smaller reductions in food and water intakes and body weight, without change in sodium intake. In separate experiments, Fos immunoreactivity was increased in several brain nuclei known to be involved in the control of body fluid and energy homeostasis, e.g. central nucleus of the amygdala, median preoptic nucleus, bed nucleus of the stria terminalis and arcuate nucleus. Increased Fos expression was similar for ICV and SC infusions when measured on days 2–3 or 6–7 of infusion. In conclusion, increases of brain activity by UCN may be associated with stimulation of adrenocorticotrophic hormone release and sympathetic nervous activity, but increases may also indicate suppression of ingestive behaviours by stimulating central inhibitory mechanisms located in areas known to control body fluid and energy homeostasis.

Water and sodium intake of wild and New Zealand rabbits following angiotensin

Two rabbit strains, New Zealand (laboratory) rabbits and Australian wild rabbits, both members of the Oryctolagus cuniculus genus were studied. New Zealand rabbits under control conditions consumed 2-5 times more water and 8-30 times more 0.5 M NaCl/kg body weight than wild rabbits. Single injections of angiotensin II or III administered ICV did not induce water drinking in either strain. Acute ICV infusion of angiotensin II also did not influence water intake, but after several days of administration, induced increased sodium intake. Intravenous infusion of graded doses of angiotensin II induced diuresis only at the higher doses in both strains. In New Zealand rabbits, this was accompanied by a commensurate and concurrent increase in water intake. Intravenous infusion of angiotensin II also induced urinary sodium loss that was either accompanied or followed by increased sodium intake. The development of salt appetite in both strains was preceded by sodium loss.

Sodium Intake and Reproduction in BALB/C Mice

The effect of sodium intake on the reproductive performance of BALB/C mice was assessed in four groups of 11 or 12 mice that received ad lib access to low or higher sodium food (LSF 4-5, HSF 120-143 mmol Na+/kg). The two groups that received HSF had (mean values) 100% matings, 83 and 91% litters, 5.9 pups/litter, pups weighing 2.05 and 2.22 g (3 days after birth) and 10.47 and 10.96 g at weaning (19 days). One of the HSF groups that also had 300 mM NaCl to drink did not show any benefit. Two groups received LSF, and one of them also received 30 mM NaCl. The group given LSF only had 83% matings, 20% litters, 1.5 pups/litter, and pups that were significantly smaller at birth and at weaning. However, the LSF group given 30 mM NaCl to drink performed almost as well as the two HSF groups. The results show that (a) the daily sodium requirement for optimal reproduction was > or = 400 (micromol/day, based on voluntary sodium intake late in gestation and lactation; (b) sodium deficiency was the cause of reproductive deficiency in mice on LSF; (c) severe sodium deficiency suppressed reproduction primarily at the gestation step; (d) this deficiency could be prevented by the voluntary sodium intake of mothers with access to salt solution; and (e) pups on the LSF showed an avid innate salt appetite when offered salt solution at 12 days of age.

Angiotensin II stimulates intake of ethanol in C57BL/6J mice.

The influence of intracerebroventricular (i.c.v.) infusion of angiotensin II on intake of water and ethanol solutions was determined in C57BL/6J mice. Compared to other mice, C57 mice do not show an aversion to ethanol solutions. With both water and ethanol solutions available, the C57 mice consumed 40-60% of their total daily fluid intake as ethanol solution when the concentration of ethanol solution offered was 4-14%. When given a choice between 0.3 M KCl and either 4 or 10% ethanol solution, the mice clearly preferred the ethanol solution. With water only available, i.c.v. infusion of angiotensin II increased intake from 3-5 mL/day (baseline) to 11-12 mL/ day (Day 4 of infusion). A similar increase in intake occurred in mice with access to a nonpreferred solution of 0.3 M KCl. In comparison, when only 4% ethanol solution was available, angiotensin II increased intake to 7-8 mL/day, and when only 10% ethanol solution was available, intake was transiently increased. The results demonstrated that thirst for water caused by i.c.v. infusion of angiotensin II in C57 mice is similar to that observed in BALB/C mice. Unlike BALB/C mice, however, i.c.v. infusion of angiotensin II stimulated intake of ethanol solution. The failure of angiotensin II to cause a large increase in 4% ethanol solution or a sustained increase in 10% ethanol solution intake does not seem to be caused by an aversion to the taste of ethanol solution, but most likely due to postingestional factors.

Sodium and water intake of sheep, rabbits and cattle during ICV infusion of eledoisin

The present study reports the effects of ICV administered eledoisin, the most potent anti/dipsogenic member of the tachykinin family, in three species. Sheep with chronic parotid fistula lost daily 200-400 mmol sodium in 3-4 l of saliva. During ICV infusion of eledoisin, 2 to 50 ng/min, a decrease in sodium intake was observed. If water was withheld for 22 hours, sheep normally drank 5.4 l water on presentation. During ICV infusion of eledoisin, 50 ng/min, water intake increased significantly. Wild rabbits lost 5 mmol sodium in 50 ml of urine after injection of furosemide. During ICV infusion of eledoisin, 30 ng/min, a decrease in sodium intake and an increase in water drinking was observed. Cows prepared with parotid fistula had access to sodium solution every other day to replace salivary sodium loss. During ICV infusion of eledoisin, 50 and 150 ng/min, a decrease in sodium intake occurred, and water intake was unaffected. These results confirm that central administration of eledoisin specifically influences ingestive behaviour in mammals and draws attention to some species differences in the observed effects.

Central action of basic fibroblast growth factor on ingestive behaviour in mice

Intracerebroventricular (ICV) infusion of basic fibroblast growth factor (FGF-2) at 50 ng/h for 5 days in male BALB/c mice suppressed the daily intakes of water and food (n = 4). Intakes were reduced on the second day, and were suppressed until the second day after stopping the infusion. The same infusion for 4 days had little effect on the high intakes of 0.3 M NaCl solution and water induced by prolonged ICV infusion of angiotensin II, or the daily food intake in these experiments (n = 7). However, the same infusion for 3-4 days reduced the increased intake of NaCl solution in Na-depleted mice (n = 8), reduced the increased water intake of water-restricted mice (n = 6 or n = 7), and reduced daily food intake in both experiments. Ventricular enlargement was noted in mice at the end of these experiments but, for reasons advanced, did not appear to account for the responses. The results indicate that FGF-2 may have an inhibitory role in these ingestive behaviours.