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When hypothyroidism is induced surgically in early steps of development in the rat, an increase in serum aldosterone concentration (AC), in absence of changes in plasma renin activity (PRA), is observed. In contrast, in propylthiouracil (PTU) induced hypothyroidism, in adult animals, both AC and PRA decrease. Potassium iodide (KI) or triiodo-L-thyronine (T3) administration to thyroidectomized rats restores AC to normal levels, increasing PRA during the latter treatment. A close relationship between AC and plasma renin concentration (PRC) is observed in these experimental situations. The decrease in urinary aldosterone concentration (ACu), and the relation found between AC/ACu ratio and T3 concentration, suggest that metabolic clearance of aldosterone might be related to peripheric T3 levels in thyroidectomized animals, treated with KI or T3. These observations support the hypothesis, previously reported, which suggests different mechanisms involved in the control of aldosterone and renin release during the two different types of hypothyroidism.

In segments of isolated proximal colon from bilaterally adrenalectomized and nephrectomized rats 10−12 g angiotensin/ml added to the serosal medium stimulated fluid transfer and sodium transport whose increase was accompanied by a decrease in transepithelial potential difference and short circuit current. Transepithelial electrical resistance remained unchanged. Replacement of chloride by sulphate in the bathing medium blocked the response to angiotensin. This suggested that angiotensin stimulated a coupled NaCl transport. Administration of aldosterone also blocked the response to angiotensin, suggesting that a low concentration of endogenous aldosterone is partly responsible for the action of angiotensin. This would explain the necessity for some form of pretreatment such as bilateral adrenalectomy and nephrectomy. Hydraulic conductivity of proximal colon sacs was computed from the variation in the water flow from serosa to mucosa in response to an osmotic shock. A significant (P < 0·01) increase in the hydraulic conductivity of the proximal colon of bilaterally adrenalectomized and nephrectomized rats was found when 10−12 g angiotensin/ml was present in the serosal bathing medium. The observed decrease in the transepithelial potential difference has been related to an action of angiotensin on the paracellular shunt pathway.

The basal levels of angiotensin I and kinetic parameters of renin-angiotensin system were studied under control, hyper- and hypothyroidism conditions. The serum levels of triiodothyronine (T3) and thyroxine (T4) and plasma angiotensin II have been measured radioimmunoassay. Hyperthyroidism was induced by 5.5 micrograms/200 g of T3 or 100 micrograms/200 g of T4 administration for 12 days, and hypothyroidism by propylthiouracil (PTU) administration of 1 mg/200 g for 12 days. Basal levels of angiotensin I and plasma renin activity (PRA) increased after T3 injection, were not altered by T4 and decreased after PTU administration. T3 and T4 induced an increase in plasma renin concentration (PRC), while PTU induced a decrease in PRC. Plasma renin substrate (PRS) decreased in hyperthyroid rats and was unchanged by experimentally induced hypothyroidism. A good correlation between T3 serum levels and PRA was found, but there was no such correlation between T4 serum levels and PRA.

The relationship between the renal function and some components of the renin-angiotensin system has been studied in hypothyroid rats thyroidectomized surgically at different periods of their life. Changes in plasma renin concentration (PRC) depending on the period hypothyroidism were induced. Results showed that the renin release control could result from an equilibrium between the reduced beta-adrenergic activity and the marked natriuresis observed in hypothyroidism. A reduction in plasma angiotensinogen concentration (PAC), due to a decrease in its hepatic production, was observed in thyroidectomized animals. PAC reduction was independent of the hypothyroidism induction period. Alterations in plasma renin activity (PRA) were a consequence of PRC and PAC changes in thyroidectomized animals, as an increase in fractional sodium excretion (FENa) time course dependent, was found in these rats.

The effects of endogenous Plasma Renin Substrate (PRS) on the relationship between Plasma Renin Activity (PRA) and the Plasma Renin Concentration (PRC) have been studied in hyperthyroid rats, by I-triiodothyronine (T3) administration and in hypothyroid rats, by propylthiouracil (PTU) treatment, to clarify if PRA changes are an adequate index for evaluating the renin-angiotensin changes during the alterations in the thyroid function. Although in experimental situations studied the induced variation on PRC explains a 62 per cent of the changes in PRA, finding a good lineal correlation between both parameters (r = 0.79, P less than 0.001). Not only does PRS play an important role on the kinetic of the enzymatic reaction but also explains jointly with PRC up to a 85 per cent of PRA alterations. PRS changes become more important during thyrotoxicosis where they limit in a higher degree the velocity of reaction due to inverse relationship between PRC and PRS (r = 0.74, P less than 0.001).

The basal levels of angiotensin I (A. I), plasma renin activity (PRA), plasma renin substrate (PRS) and plasma renin concentration (PRC) were estimated in hyperthyroid rats before and after propranolol treatment. Serum levels of triiodothyronine (T3), thyroxine (T4), angiotensin I both basal and generated by incubation of plasma samples were measured by radioimmunoassay, while, PRC and PRS were evaluated by triiodothyronine or thyroxine treatment induced a decrease in PRS and an increase in PRC whereas triiodothyronine alone produced an increase in the basal levels of angiotensin I and PRA. The propranolol in euthyroid animals not only decreased the basal levels of angiotensin I and PRA, but also prevented the PRC increase in hyperthyroid rats, while restoring at the same time the basal levels of angiotensin I and PRA.

A new spectrofluorimetric method for protein estimation has been developed and applied to study the renin-angiotensin system. The fluorometric reagent, 1-4-diamino-2-3-dichloro-anthraquinone, is introduced in this field and used for the first time. Renal semipurified rat renin is incubated with a synthetic substrate (N-acetyl-tetra-decapeptide) at 0 degrees C and 37 degrees C, at optimal pH, during 3 hours. The incubated mixture is studied by bioassay (BA), radioimmunoassay (RIA) and spectrofluorimetric analysis (SFA), and values obtained with these methods are compared. Blank samples for spectrofluorimetric analysis were prepared by substituting the incubated mixture with the unincubated components of the reaction. Its fluorescence values were subtracted from those of the incubated mixture. Precision and sensitivity for RIA and SFA were similar in both cases, but different in the case of BA. Renal renin activity (RRA) values for RIA (7.15 x 10-(3) nM/ml angio. I/3 h) and SFA (7.08 x 10-(3) nM/ml angio. I/h) were statistically equal (t = 1.05; p less than 0.05), while RRA values for BA (6.23 x 10-(2) nM/ml angio. I/3 h), were higher and significantly different from the statistical point of view. The graphical representation of RRA values for RIA versus SFA values gives an objective expression where RRA(RIA) = 0.78 RRA(SFA) + 0.02.

SummaryWe report the genetic abnormalities in the protein C genes of a Spanish child with neonatal purpura fulminans and disseminated intravascular coagulation, associated with undetectable protein C levels. Direct sequencing of the nine protein C gene exons and their splice junctions indicated that the proband is a compound heterozygote with two mutant protein C gene alleles, Y124C and Q132X, that do not express protein C in plasma. The Y124C mutation was inherited from the mother and is due to a novel A to G transition at nucleotide 3416, which results in the substitution of cysteine for tyrosine 124, a highly conserved amino acid in EGF-like domains. The paternal inherited mutation (Q132X) is a C to T transition at nucleotide 3439, which replaces glutamine 132 with a Stop codon signal. This mutation, if expressed, should result in the synthesis of a truncated protein of 131 amino acids. Y124C or Q132X are present in the heterozygous state in the asymptomatic parents and siblings of the proband, all of which have half the normal plasma levels of protein C. Q123X has also been identified in families where type I PC deficiency is inherited as a clinically dominant trait. Therefore, the presence of the same mutation in a family showing a clinically recessive pattern of inheritance indicates that other factors, apart from the type of protein C gene mutation, are responsible for the clinical expression of protein C deficiency.

To elucidate the molecular basis of hereditary protein S (PS) deficiency and, in particular, type III or free PS deficiency, the allelic distribution and segregation patterns of the PS gene (PROS1) polymorphisms P626A/G and S460P (PS Heerlen) have been analyzed in a group of 45 proposita suffering from type I or type III PS deficiency. No differences between patients and controls were found in the frequency of the P626A/G alleles. By contrast, the frequency of the PS Heerlen allele in the group of patients with type III PS deficiency (9 of 46 chromosomes, P = .196) was significantly higher (P.001) than in the control group (1 of 300 chromosomes, P = .003). The A allele of P626A/G was always associated with the P allele of S460P. However, this haplotype did not co-segregate with the type III PS-deficient phenotype in 3 of the families. Furthermore, multipoint linkage analysis excluded the whole PROS1 gene in 1 of these families, which is in agreement with the absence of mutations in the PROS1 gene, as determined by sequence analysis. Finally, linkage analysis with 4 microsatellite markers linked to the C4BPB and C4BPA loci also excluded these two genes. From these results we conclude that, at least in some families, the molecular basis of type III PS deficiency is not due to the Mendelian inheritance of a single defect in the PROS1 or in the C4BP genes.

The changes occurring in several components of the rat renin-angiotensin system (RAS) were studied for the brief postnatal period, between the fourth and tenth week of life. The parameters were: plasma renin activity (PRA), plasma renin concentration (PRC), plasma renin substrate (PRS) and the plasma angiotensin II concentration (AII). A gradual decrease in PRA with age was noticed. Between the fourth and the eighth weeks of life, this was attributed to a corresponding decline in both PRC and PRS. However, between the eighth and tenth weeks, no changes in PRA could be detected, but PRC and PRS increased, perhaps as a consequence of the changes in renal function and the AII increase observed. In this second period, simultaneously with the RAS changes described, there was reduced sodium chloride excretion as the glomerular filtration rate (GFR) stabilized. The data presented suggest that this postnatal period is critical, in rats, for the maturation of the RAS component control mechanisms; they appear to be closely related to the development of the renal function.