Measurement of extracellular fluid volume in the neonate using multiple frequency bio-impedance analysis.

Bioelectrical impedance analysis may be useful for cot-side monitoring of fluid balance in the neonatal intensive care unit (NICU). However the presence of cardio-respiratory monitoring equipment, non-ideal electrode placement and inability to obtain accurate crown-heel measurements may interfere with the ability to obtain reliable impedance data in this setting. This study aimed to investigate the effects of these factors on impedance analysis and to develop a prediction equation for extracellular fluid volume in the neonate. The study found that cardio respiratory monitoring had no significant effect on multi-frequency impedance measurements. Placement of current delivering electrodes on the ventral rather than dorsal surfaces improved separation of current and voltage electrodes but did not alter impedance results. Contralateral measurements were not significantly different to ipsilateral measurements. In 24 infants, extracellular fluid volume was measured using corrected bromide space (CBS) and simultaneous impedance analysis was performed. There was good correlation between CBS and the impedance quotient FF2/Ro where F is foot length and R0 is resistance at zero frequency. The study concludes that despite many potential difficulties associated with impedance analysis in the NICU, reliable measurements of impedance can be obtained and further work to validate prediction equations for ECF is warranted.

Effect of steroid hormones on blood pressure.

1. There is considerable evidence to support the idea that steroid hormones have the potential to increase blood pressure that may not always be via 'classical' mineralocorticoid or glucocorticoid action. 2. Epidemiological studies, together with the evidence from studies in animals, proposed the link between an adverse intra-uterine environment (i.e. undernutrition or excess exposure to glucocorticoids) and the early onset of cardiovascular and metabolic diseases later in life. 3. We tested this by treating pregnant ewes (and foetuses) with excess steroid early in pregnancy. The mean ages at which the prenatal exposure to glucocorticoid (dexamethasone 0.48 mg/h for 48 h) occurred were 22 +/- 0.4 to 29 +/- 0.4 days (prenatal treatment group 1; PTG1) and 59 +/- 2 to 66 +/- 2 days (PTG2), respectively. Basal blood pressures and hormones and the vascular responsiveness to graded doses of angiotensin II and noradrenaline, or to a 5-day adrenocorticotropin hormone treatment (ACTH), in lambs at 4, 10 and 19 months of age were studied. 4. Basal mean arterial pressure in PTG1 group (80 +/- 1 mmHg at 4 months; 83 +/- 1 mmHg at 10 months; and 89 +/- 1 mmHg at 19 months; n = 6) was significantly different (P < 0.05 in all groups) from that in the control group of lambs (74 +/- 2 mmHg at 4 months; 76 +/- 1 mmHg at 10 months; and 81 +/- 1 mmHg at 19 months; n = 7). Prenatal glucocorticoid exposure did not alter vascular responsiveness to noradrenaline, angiotensin II and ACTH in these sheep at any of the ages studied. 5. These results suggest that foetal exposure to maternal dexamethasone during defined developmental stage or 'window' programmes elevated blood pressure, which persists later in life.

Altered cardiovascular haemodynamics and baroreceptor-heart rate reflex in adult sheep after prenatal exposure to dexamethasone.

Numerous epidemiological studies, together with mounting evidence from studies in animals, point to a correlation between an adverse intrauterine environment and the early onset of cardiovascular and metabolic diseases later in life. We were the first to show that sheep exposed to dexamethasone (0.28 mg.kg-1.day-1 for only 2 days) at the end of the first month of pregnancy (PTG1), but not those exposed at the end of the second month of pregnancy (PTG2), had a higher basal mean arterial pressure (MAP) 19 months after birth. In the present study we report the MAP, cardiovascular haemodynamics and baroreflex sensitivity in these animals at 40 months of age. MAP in the PTG1 group was significantly higher than in the control group (91+/-1 mmHg and 81+/-1 mmHg respectively; P<0.001) and also when compared with the PTG2 group (82+/-1 mmHg; P<0.001). There was a significant increase in cardiac output in the PTG1 group compared with the control group (108+/-2 and 96+/-4 ml.min-1.kg-1 respectively; P<0.05). The increase in cardiac output in the PTG1 group was due to an increase in stroke volume (1.82+/-0.08 ml.kg-1. beat-1, compared with 1.46+/-0.06 ml.kg-1.beat-1 in the control group; P<0.05), but not in heart rate. In the hypertensive group of animals (PTG1), there was a rightward shift of the baroreflex curve. In group PTG2 (the normotensive group of animals), a lower gain was found before and during propranolol treatment. The decrease in gain of the baroreflex was not associated with changes in heart rate range, suggesting an impairment in the central processing of the baroreceptor signals. Thus sheep fetuses exposed to dexamethasone for only 2 days at the end of the first month of gestation have high blood pressure (dependent upon the increase in cardiac output) and a reset of the baroreflex at 40 months of age. Animals that have received prenatal dexamethasone closer to mid-gestation, although normotensive with normal cardiac output, showed an altered baroreceptor-heart rate response.

Prediction of aminoglycoside distribution space in neonates by multiple frequency bioelectrical impedance analysis.

OBJECTIVE: Aminoglycoside antibiotics have a narrow margin of safety between therapeutic and toxic levels. The current study used multiple frequency bioelectrical impedance analysis to develop prediction equations for gentamicin distribution space in neonates. METHODS: Gentamicin pharmacokinetic parameters and bioimpedance were measured in 14 infants in the neonatal intensive care unit. Stepwise regression analysis was used to develop predictive models, using impedance quotients (F2/R), weight and gestational age as variables, whose predictive performance was then tested in a second group of ten infants. RESULTS: The prediction model with the smallest bias and highest concordance correlation was that which included F2/R0 and weight. This bias of 50 ml or 6.7% was less than half of that found using a model including weight alone. CONCLUSION: A bioelectrical impedance-based prediction equation for prediction of gentamicin distribution space in neonates was produced. Although this prediction equation represents only a small improvement over that using weight alone, this is of clinical significance due to the narrow margin between therapeutic and toxic levels for gentamicin. A clinical trial to confirm the value of this methodology is now warranted.

Late steps of aldosterone biosynthesis: sheep are not rats.

1. The last three steps of aldosterone biosynthesis have been demonstrated to be catalysed by a single enzyme, referred to as CYP11B (or P450(11) beta) in cow, pig, sheep and bullfrog and as CYP11B2 (or P450aldo) in rat, human, mouse and hamster. 2. The related enzyme CYP11B1 (also referred to as P450(11) beta) in rat, human, mouse and hamster does not have aldosterone synthesis activity, but no such enzyme has been reported in the cow, pig or sheep to date. 3. Exclusive aldosterone secretion in the zona glomerulosa (ZG) of the adrenal cortex in species such as rat, human, mouse and hamster could be ascribed to the restricted distribution of CYP11B2 to the same region in the adrenal cortex. 4. In other species, such as cow, pig and sheep, the CYP11B enzyme is expressed throughout the adrenal cortex and, thus, the exclusive aldosterone biosynthesis in the ZG could not be explained simply by the distribution of the enzyme. 5. We have shown in the sheep that potassium loading and acute sodium depletion stimulate the CYP11B transcript levels, which are not further increased by chronic sodium depletion. 6. The predominant CYP11B in the sheep adrenal cortex catalyses the synthesis of aldosterone from deoxycorticosterone (DOC) in vitro, is expressed throughout the adrenal cortex and the corresponding transcript levels are increased by K+ loading or sodium depletion. In short, as far as the last step of aldosterone biosynthesis is concerned, sheep are different from rats. In the rat, the CYP11B2 transcript or protein is elevated by K+ loading or sodium depletion, but not the CYP11B1 transcript or protein. 7. We propose that during severe sodium deficiency there is a switch in the aldosterone pathway to one preferentially involving 18-OH-DOC and not corticosterone.

Location of ganglioside GM2 activator protein gene expression in sheep.

1. The present study was aimed at characterizing and establishing the site of production of a 'novel' protein isolated in 1988 during the course of studies on sheep renal morphology. This protein has subsequently been identified as the GM2 activator protein (GM2AP). 2. The 'novel' protein, with an apparent molecular weight of 18-22 kDa and a pI between 4.7 and 4.9, was isolated from enriched granular fractions of sheep kidney cortex using two-dimensional (2-D) polyacrylamide gel electrophoresis. Following electroelution, the N-terminal amino acid sequence was determined and, applying the preferred codon usage formula, an oligodeoxyribonucleotide probe was constructed for examination of sites of expression of this novel protein using northern analyses and hybridization histochemistry. 3. Western blots of the 2-D gels onto nitrocellulose membranes permitted us to select the appropriate spots for injection into rabbits for production of polyclonal antibodies. The antibodies were used to confirm the sites of protein production using immunohistochemistry. 4. Northern analyses revealed that GM2AP mRNA has a widespread distribution in ovine tissues. In the kidney, GM2 was expressed in all major renal arteries and arterioles. In the liver, the expression of the gene was prominent in the hepatic vein and ducts. Antibodies raised against the GM2AP confirmed that the protein was present at the same sites as the mRNA. 5. These are the first studies showing the location of GM2 activator gene expression in normal mammalian tissues. The arterial site of production has implications for local action or an important role in membrane integrity throughout the kidney.

Effects of relaxin, pregnancy and parturition on collagen metabolism in the rat pubic symphysis.

The aim of this study was to examine the changes in collagen metabolism that occur during pregnancy and parturition and upon relaxin administration to the rat pubic symphysial interpubic tissue. Pubic symphyses were collected from non-pregnant, and intact and ovariectomised pregnant Sprague-Dawley rats at days 15, 18 and 21 of pregnancy as well as during and after delivery, and analysed for collagen content and solubility. SDS-PAGE was used to determine collagen composition. During pregnancy and particularly during birth, there was a significant reduction in both the tissue wet (57+/-3%) and dry (43+/-3%) weight (n=7), which coincided with a significant increase in water content (to 80%) and was attributed to a significant (P<0.05) reduction in overall tissue collagen content (by 47+/-2%). This resulted in both soluble (10%) and insoluble (90%) collagen levels being reduced, but gel electrophoresis demonstrated the presence of types I, II and V collagen in all samples. Western blot analysis confirmed the presence of type II collagen throughout pregnancy, confirming that the rat pubic symphysis remained a fibrocartilaginous tissue throughout gestation. In the absence of the ovaries and hence relaxin, tissue collagen content and solubility were not significantly different from control measurements. However, tissues of ovariectomised animals treated with oestrogen and progesterone (pellets) and relaxin (injection) contained collagen levels that mimicked those of late pregnancy and parturition. These results suggest that relaxin plays an important role in regulating collagen catabolism during gestation in the rat.

Hypothesis: aldosterone is synthesized by an alternative pathway during severe sodium depletion. 'A new wine in an old bottle'.

1. The last three steps of aldosterone biosynthesis, 11 beta-hydroxylation, 18-hydroxylation and 18-oxidation, have been demonstrated to be catalysed by one enzyme, which is the cytochrome P450(11 beta) (CYP11B) in cow, pig, sheep and bullfrog or cytochrome P450aldo (CYP11B2) in rat, human, mouse and hamster. 2. The related enzyme P450(11 beta) (CYP11B1) from rat, human, mouse and hamster adrenals displays 11 beta-hydroxylation and 18-hydroxylation activities, but not 18-oxidation activity in vitro. No such enzyme has been reported in the cow, pig or sheep to date. 3. Data showing the dissociation of aldosterone secretion from plasma angiotensin II (AngII) levels indicate the presence of other factor(s) that regulate aldosterone biosynthesis in response to changes in body sodium status. Thus, we propose the existence of a 'sodium status factor' that regulates aldosterone biosynthesis in addition to AngII, K+, adrenocorticotropic hormone and atrial natriuretic peptide. 4. We propose that during severe sodium deficiency there is a switch in the aldosterone pathway to a pathway using 18-hydroxy-deoxycorticosterone (18-OH-DOC) rather than corticosterone as an intermediate. This switch may be mediated via the putative 'sodium status factor'. 5. Two models of the hypothesis will be discussed in this paper: (i) a 'one-enzyme' model; and (ii) a 'two-enzyme' model. 6. The one-enzyme model proposes that P450aldo (P450(11 beta) as in the case of the cow, sheep and pig) changes its enzymatic activity during severe sodium deficiency (i.e. switching to the alternative aldosterone biosynthesis pathway). 7. The two-enzyme model proposes that, under normal circumstances, P450aldo synthesizes aldosterone from deoxycorticosterone, while during severe sodium deficiency the P450(11 beta) provides the substrate (i.e. 18-OH-DOC) for the P450aldo.

An early prenatal exposure to excess glucocorticoid leads to hypertensive offspring in sheep.

1. Recent studies in animals have linked fetal exposure to excess maternal glucocorticoids with the later occurrence of cardiovascular disorders, particularly hypertension. 2. To test the hypothesis that prenatal treatment could impact on adult blood pressure two groups of pregnant ewes were transported from the farm to the Institute at either 22-29 days of pregnancy (pretreatment group 1) or 59-66 days of pregnancy (pretreatment group 2), subjected to 48 h treatment with dexamethasone (0.28 mg day-1 kg-1 for 2 days) and then returned to the farm. The control group remained at the farm for the entire pregnancy. Lambs were then studied at approximately 4, 10 and 19 months after birth. 3. The basal mean arterial pressure in pretreatment group 1 (80 +/- 1 mmHg at 124 days; 83 +/- 1 mmHg at 309 days and 89 +/- 1 mmHg at 558 days; n = 6) was significantly different (P < 0.05 in all groups) from that in the control group of lambs (74 +/- 2 mmHg at 110 days; 76 +/- 1 mmHg at 323 days and 81 +/- 1 mmHg at 568 days; n = 7). However, prenatal glucocorticoid exposure did not alter vascular sensitivity to noradrenaline, angiotensin II and adrenocorticotropic hormone in these sheep at any of the ages studied, nor did it affect basal or adrenocorticotropic hormone-induced concentrations of cortisol or basal plasma renin concentrations in the lambs at any age. 4. These data support the hypothesis that excess glucocorticoid exposure in early pregnancy, during a critical developmental stage or 'window', programmes higher blood pressure that persists in later life.

Carbenoxolone does not cause a syndrome of mineralocorticoid excess in sheep.

These studies investigated whether treatment with carbenoxolone (CBX), an inhibitor of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD), resulted in an enhanced mineralocorticoid response to endogenous or infused cortisol. In conscious sodium replete sheep with a parotid fistula, infusion of CBX (40 mg/h for 10 days) did not increase mean arterial pressure, or change sodium and potassium status or plasma renin concentration, but significantly increased the half-life of 1,2[3H] cortisol from 18.6 +/- 4.0 to 38.8 +/- 3.9 min (p < 0.05) and reduced the blood clearance rate of cortisol (BCR) from 31 +/- 3 to 15 +/- 4 L/h (p < 0.01). The reduction in cortisol BCR was associated with reduction in cortisol secretion rate from 433 +/- 116 to 181 +/- 79 nmol/h (p < 0.01). Cortisol (8 mg/h) for 5 days increased mean arterial pressure (from 83 +/- 2 to 101 +/- 5 mmHg, p < 0.001) and caused natriuresis, hypokalaemia and hyperglycaemia. These responses were unaltered when cortisol was infused from the fifth to the tenth day of CBX infusion. These findings suggest that in sheep, carbenoxolone is either a less potent inhibitor of 11 beta-HSD2 than in other species or 11 beta-HSD2 may not be the only mechanism, which determines the specificity of the MR.

Structure of an ovine CYP11B1 gene.

Glucocorticoids play an important role in the normal development and proliferation of cells, and are also involved in inflammatory responses. The level of active glucocorticoids in the body is controlled in part by the enzyme CYP11B1, which catalyses the final step of its biosynthesis. In this report, we have completely characterised the ovine CYP11B1 gene using two overlapping clones isolated from an lambdaEMBL3 sheep liver genomic library. The gene comprised 9 exons and 8 introns, spanning over a region of 8.0 kb. Two ovine CYP11B1 transcripts, with molecular sizes of 1.9 and 4.0 kb, have also been isolated from the adrenal zona fasciculata region, which showed that they arose from the usage of the two polyadenylation sites situated 2.1 kb apart in exon 9. The transcriptional start sites of the gene has been mapped using primer extension analysis. Three major start sites were identified at positions -5, -6 and -77 from the first ATG codon (Met), with two minor sites located at positions -306 and -413. When examined in context with the ovine CYP11B1 5' regulatory region, the results suggested that the ovine CYP11B1 gene contained two additional core promoters located further upstream of a proximal TATA box which could be utilised to produce mRNAs with alternative transcriptional start sites.

Effects of angiotensinogen antisense oligonucleotides on fluid intake in response to different dipsogenic stimuli in the rat.

The role of centrally synthesised angiotensinogen in neural mechanisms subserving water drinking in rats was investigated by injecting antisense oligonucleotides complementary to rat angiotensinogen mRNA into the brain with the aim of inhibiting cerebral angiotensinogen synthesis. Phosphorothioate antisense oligonucleotides (18 mer) encompassing the translation start codon were injected into the lateral ventricle of rats and their responses to a number of dipsogenic stimuli tested. These were: intracerebroventricular (i.c.v.) renin, i.c.v. angiotensin II, i.c.v. carbachol, subcutaneous isoproterenol, intravenous hypertonic saline, water deprivation for 24 h or subcutaneous injection of polyethylene glycol. Antisense treatment significantly reduced (by approximately 50%) the volume of water drunk in response to i.c.v. injection of renin or subcutaneous isoproterenol, but did not reduce water intake elicited by the other dipsogenic stimuli. The i.c.v. administration of mismatch, scrambled or sense oligonucleotides did not inhibit water intake. These data suggest that centrally produced angiotensinogen may have a role in neural pathways subserving isoproterenol-induced drinking.

Angiotensinogen antisense oligonucleotides and fluid intake.

The effectiveness of antisense oligonucleotides (ODNs) to angiotensinogen on intracerebrovenricularly injected renin induced thirst was investigated. As a corollary, information would be gained about the role of centrally synthesised angiotensinogen in the neural mechanisms subserving water drinking in rats. Stable, easily synthesised phosphorothioate antisense oligonucleotides (18 mer), one of which included the sequence encompassing the translation start site, were injected into the lateral ventricle of rats. The drinking response to a number of dipsogenic stimuli was tested. Antisense significantly reduced (by about 50%) the volume of water drunk in response to intracerebroventricular (icv) renin or isoproterenol but did not reduce drinking in response to the physiological challenge of icv angiotensin II, icv carbachol, intravenous hypertonic saline, water deprivation or subcutaneous injection of polyethylene glycol. Only one out of four antisense probes gave positive results, while mismatch or scrambled oligonucleotides did not inhibit water intake. This finding reduces the probability that the results observed are non-specific. In these experiments, an ODN specific for angiotensinogen was discovered and was produced easily in large enough amounts and stabilised against intracellular nucleases without floss of cellular access or biological effect.

Redistribution of carbonic anhydrase VI expression from ducts to acini during development of ovine parotid and submandibular glands.

Carbonic anhydrase VI (CA VI) is a secreted enzyme produced predominantly by serous acinar cells of submandibular and parotid glands. We have investigated the developmental pattern of CA VI production by these glands in the sheep, from fetal life to adulthood, using immunohistochemistry. Also, a specific radioimmunoassay for CA VI was used to measure changes in enzyme expression in the parotid gland postnatally. CA VI is detectable by immunohistochemistry in parotid excretory ducts from 106 days gestation (term is 145 days), in striated ducts from 138 days and in acinar cells from 1 day postnatal. The duct cell content of CA VI declined as the acinar cell population increased, a feature also of CA VI immunoreactivity in the submandibular gland. Production of CA VI by submandibular duct cells was detectable initially at 125 days gestation, and acinar production was not seen before 29 days post-natal. Apart from the differing ontogeny of CA VI production in ducts and acini of parotid and submandibular glands, there was a parallel pattern of CA VI expression during the development of these major salivary glands. With the development of the acinar tissues in the postnatal lamb, there was a dramatic increase (about 600-fold) in the level of expression of CA VI in the parotid gland between days 7 and 59 as measured by radioimmunoassay.

Investigation of the mineralocorticoid and hypertensinogenic activity of 18-hydroxycortisol in conscious sheep.

The increased urinary excretion of 18-hydroxycortisol (18-OHF) in patients with primary aldosteronism has raised the possibility that 18-OHF is involved in the maintenance and/or pathogenesis of the associated hypertension. This study has investigated the mineralocorticoid, glucocorticoid, and hypertensinogenic activities of 18-OHF in the conscious sheep. Infusion of 18-OHF (400 micrograms/h i.v. 5 days; n = 5) alone had no effect on blood pressure or on fluid and electrolyte balance. Infusion of a combination of five adrenal steroids (aldosterone 3 micrograms/h, cortisol 5 mg/h, corticosterone 0.5 mg/h, 11-deoxycortisol 1 mg/h and deoxycorticosterone 25 micrograms/h, i.v. 5 days; n = 5) increased blood pressure by 14 +/- 1 mmHg (p < .001), but when 18-OHF was infused together with the five adrenal steroids, no additional increase in blood pressure was observed. In another group of sheep (n = 4) 18-OHF was infused at a range of doses (5, 50, 100, 200, 500, and 1000 micrograms/h i.v.), each for 2 h, into sodium-replete and sodium-deplete, adrenalectomized sheep. 18-OHF had no effect on the urinary sodium or potassium excretion or on the salivary Na/K ratio in either group as compared with vehicle infusion. To examine the renal effects of 18-OHF, a range of doses of 18-OHF (5, 50, 100, 200, 500, and 1000 micrograms/h) were infused directly into the renal artery of conscious sheep (n = 4). 18-OHF did not affect the renal blood flow nor the urinary sodium or potassium excretion compared with vehicle infusion. In summary, we could not demonstrate any mineralocorticoid, glucocorticoid, or hypertensiongenic effects of 18-OHF in conscious sheep at a dose of 400 micrograms/h. Thus, a cautious approach to interpreting the role that 18-OHF plays in the clinical manifestations of primary aldosteronism, is necessary.

Aldosterone secretion: a molecular perspective.

The major mineralocorticoid hormone aldosterone is secreted from the zona glomerulosa of the adrenal cortex. Aldosterone is synthesized from cholesterol via a series of hydroxylations and oxidations. The enzymes involved in these reactions are mostly members of the cytochrome P450 superfamily. The final steps of this pathway, the conversion of 11-deoxycorticosterone (DOC) to aldosterone, require conversion via the intermediates 18-hydroxy-DOC or corticosterone and 18-hydroxycorticosterone. There are significant differences between species in the number of genes that encode the P450(11beta)-related enzymes (CYP11B) involved in these steps and the zonal distribution of their expression. One enzyme is capable of 11-hydroxylation, 18-hydroxylation, and 18-oxidation of DOC to aldosterone. The genetic basis of four diseases-congenital adrenal hyperplasia due to 11beta-hydroxylase deficiency, glucocorticoid-remediable aldosteronism, aldosterone synthase deficiency type I and type II-is explicable by mutations in these cytochrome P450(11beta)-related genes.

Functional and expression analysis of ovine steroid 11 beta-hydroxylase (cytochrome P450 11 beta).

In this study, the ovine steroid 11 beta-hydroxylase (P450(11 beta) or CYP11B) cDNA previously reported by us (1) was transfected into COS-7 cells. Using 3H-11-deoxycorticosterone (3H-DOC) as the substrate, and paper partition chromatography for separation of steroid products, the expressed enzyme was shown to catalyse the conversion of DOC to corticosterone (B), 18-hydroxy-11-deoxycorticosterone (18-OH-DOC), 18-hydroxy-corticosterone (18-OH-B), and aldosterone (ALDO). These results suggest that the expressed ovine cDNA exhibited 11 beta-hydroxylase, 18-hydroxylase and aldosterone synthesis activities. The enzymatic activity of the enzyme was further analysed by adding unlabelled steroids to compete with 3H-DOC. The conversion of 3H-DOC to 3H-ALDO was inhibited by the addition of excess DOC, B and 18-OH-DOC, indicating that all these steroids were potential substrates of the enzyme. The results also demonstrated that 18-hydroxylation could occur before 11 beta-hydroxylation with this enzyme. However, the addition of excess cold 18-OH-B had no significant effect on the level of 3H-ALDO that was synthesised. This result could imply that 18-OH-B is not an intermediate involved in the conversion of DOC to aldosterone, or, more likely, the enzyme substrate site is not accessible readily. Our results also indicated that DOC was preferred to 18-OH-DOC as a substrate for the enzyme. We have demonstrated by hybridisation histochemistry using specific oligonucleotide probes that the corresponding P450(11 beta) RNA transcript was present in all zones in the sheep adrenal cortex. In summary, we have shown that the enzyme encoded by the predominant P450(11 beta) cDNA isolated from the sheep adrenocortical cDNA library has all the enzymatic activities to biosynthesise ALDO from DOC. The corresponding transcript of this ovine P450(11 beta) cDNA was located throughout the adrenal cortex and thus the inability of the zonae fasciculata-reticularis to secrete ALDO remains to be understood.

The effect of relaxin on collagen metabolism in the nonpregnant rat pubic symphysis: the influence of estrogen and progesterone in regulating relaxin activity.

The aim of this study was to examine the effects of relaxin on collagen content, solubility, and composition in the rat pubic symphysis. Nonpregnant, female Sprague-Dawley rats were bilaterally ovariectomized and either unprimed or primed with estrogen or progesterone alone, or a combination of estrogen and progesterone. One week later these animals were given increasing doses of a synthetic human (gene-2) relaxin (0-100 micrograms) before being killed 16 h later. Their pubic symphysial tissues were then removed and analyzed for collagen content and solubility, whereas collagen composition was determined by SDS-PAGE. Relaxin administration significantly increased the length (140 +/- 6%) and weight (170 +/- 9%) of the interpubic fibrocartilage in estrogen-primed rats (n = 15). At the same time, it decreased the total collagen content by 68 +/- 6%, without altering the proportions of collagen types, which were predominantly type I (85%) and type II collagen (15%). Relaxin administered alone reduced the total collagen content by 64 +/- 4% but had no effect on collagen solubility or composition. Progesterone abolished the effects of relaxin in estrogen-primed rats. It is concluded that relaxin has a potent effect on the amount of collagen in the rat pubic symphysis that is enhanced by estrogen and antagonized by progesterone. The changes in the extracellular matrix within the pubic symphysis induced by relaxin may be important in the modifications that this tissue undergoes during pregnancy.

Evidence against a central pressor mechanism for adrenocortical steroid hypertension in sheep.

The possibility that corticotropin (ACTH)-induced hypertension results from a direct central effect of the adrenocortical steroids released by ACTH was investigated in sheep. Using two approaches, steroid levels were increased in the brain while peripheral levels remained sub-pressor. The blood pressure response to intravenous infusion of a combination of 7 steroids (aldosterone, cortisol, deoxycorticosterone, corticosterone, 11-deoxycortisol, 17 alpha hydroxyprogesterone and 17 alpha 20 alpha dihydroxyprogesterone), which causes a similar pressor effect to ACTH, was compared with that caused by intracarotid infusion of the steroids at rates calculated to give concentrations in the brain equivalent to those achieved after intravenous infusion. We also examined the effects of infusing the combination of steroids directly into the central nervous system via the lateral cerebral ventricles. Intravenous infusion of the steroids increased mean arterial pressure (MAP) from a control average of 84.0 +/- 1.1mmHg to 98.2 +/- 2.2mmHg (p < 0.001) on day 5. There was no increase in MAP during intracarotid infusion, nor during intracerebroventricular infusion. These findings suggest that the adrenocortical steroids released by ACTH do not act directly on central steroid receptors to increase blood pressure.