Effect of amiodarone on serum triiodothyronine, reverse triiodothyronine, thyroxin, and thyrotropin. A drug influencing peripheral metabolism of thyroid hormones.

2-n-Butyl-3-(4'-diethylaminoethoxy-3',5'-diiodobenzoyl)-benzofurane (amiodarone), a drug used in arrythmias and angina pectoris, contains 75 mg of organic iodine/200 mg active substance. Four studies were performed to test its effect on thyroid hormone metabolism: (a) nine male subjects were treated with 400 mg of amiodarone for 28 days; (b) five male subjects received, for the same period of time, 150 mg of iodine in the form of Lugol's solution; (c) five subjects received 300 mug L-thyroxine (T4) for 16 days; from the 10th to the 16th day, 400 mg of amiodarone was added; and (d) five euthyroid subjects received 300 mug L-T4 for 16 days. The changes in serum thyroid-stimulating hormone (TSH), serum total T4, 3,5,3'-triiodothyronine (T3), free T3, and 3,5',3'-triiodothyronine (reverse T3, rT3) were measured, and the pituitary reserve in TSH was evaluated by a thyrotropin-releasing hormone (TRH) test. The results show that amiodarone induced a decrease in serum T3 (28+/-5.1 ng/100 ml, mean+/-SEM, P less than 0.0S and 82.7+/-9.3 ng rT3/100 ml, P less than 0.01). The control study with an equal amount of inorganic iodine did not induce these opposite changes but slightly lowered serum rT3, T3, and T4. In the third study, serum rT3 increased as under amiodarone treatment, thereby proving that these changes were peripheral. It is suggested that amiodarone changes thyroid hormone metabolism, possibly by reducing deiodination of T4 to T3 and inducing a preferential production of rT3. Amiodarone also increased the response of TSH to TRH. The maximal increment of serum TSH above base line was 32+/-4.5 muU/ml under treatment and 20+/-3 muU/ml before treatment (P less than 0.01). During this test, the serum T3 increase was more pronounced than during the control period (83+/-13 and 47+/-7.4 ng/100 ml, P less than 0.05).

Identification of a major defect in insulin-resistant tissues of genetically obese (fa/fa) rats. Impaired protein kinase C.

In perfused lean rat hearts, the activator of protein kinase C phorbol myristate acetate (PMA), when present alone, stimulates glucose transport but inhibits the insulin stimulation of this transport. PMA also inactivates glycogen synthase in hepatocytes. In contrast, none of these effects are observed in hearts and hepatocytes of obese animals, indicating an impaired protein kinase C activation in these tissues, which are insulin resistant. Direct measurements of protein kinase C activity in lean rat hearts revealed that PMA provokes a translocation of the enzyme from a soluble to a particulate fraction. In obese rat hearts, the basal distribution of protein kinase C is altered (more activity is found in the soluble and less in the particulate fraction), and the translocation induced by PMA is impaired. Pretreatment of lean rats with PMA in vivo, aimed at downregulating protein kinase C, induces the same defects (i.e., insulin resistance and unresponsiveness to PMA) as those observed in hearts of untreated obese animals. The results indicate that part of the insulin resistance might be the consequence of altered modulation of insulin action by protein kinase C.

Atrial natriuretic peptide inhibits calcium-induced steroidogenic acute regulatory protein gene transcription in adrenal glomerulosa cells.

Atrial natriuretic peptide (ANP) is a potent inhibitor of mineralocorticoid synthesis induced in adrenal glomerulosa cells by physiological agonists activating the calcium messenger system, such as angiotensin II (Ang II) and potassium ion (K+). While the role of calcium in mediating Ang II- and K(+)-induced aldosterone production is clearly established, the mechanisms leading to blockade of this steroidogenic response by ANP remain obscure. We have used bovine adrenal zona glomerulosa cells in primary culture, in which an activation of the calcium messenger system was mimicked by a 2-h exposure to an intracellular high-calcium clamp. The effect of ANP was studied on the following parameters of the steroidogenic pathway: 1) pregnenolone and aldosterone production; 2) changes in cytosolic ([Ca2+]c) and mitochondrial ([Ca2+]m) Ca2+ concentrations, as assessed with targeted recombinant aequorin; 3) cholesterol content in outer mitochondrial membranes (OM), contact sites (CS), and inner membranes (IM); 4) steroidogenic acute regulatory (StAR) protein import into mitochondria by Western blot analysis; 5) StAR protein synthesis, as determined by [35S]methionine incorporation, immunoprecipitation, and SDS-PAGE; 6) StAR mRNA levels by Northern blot analysis with a StAR cDNA; 7) StAR gene transcription by nuclear run-on analysis. While clamping Ca2+ at 950 nM raised pregnenolone output 3.5-fold and aldosterone output 3-fold, ANP prevented these responses with an IC50 of 1 nM and a maximal effect of 90% inhibition at 10 nM. In contrast, ANP did not affect the [Ca2+]c or [Ca2+]m changes occurring under Ca2+ clamp or Ang II stimulation in glomerulosa cells. The accumulation of cholesterol content in CS (139.7 +/- 10.7% of control) observed under high-Ca2+ clamp was prevented by 10 nM ANP (92.4 +/- 4% of control). Similarly, while Ca2+ induced a marked accumulation of StAR protein in mitochondria of glomerulosa cells to 218 +/- 44% (n = 3) of controls, the presence of ANP led to a blockade of StAR protein mitochondrial import (113.3 +/- 15.0%). This effect was due to a complete suppression of the increased [35S]methionine incorporation into StAR protein that occurred under Ca2+ clamp (94.5 +/- 12.8% vs. 167.5 +/- 17.3%, n = 3). Furthermore, while the high-Ca2+ clamp significantly increased StAR mRNA levels to 188.5 +/- 8.4 of controls (n = 4), ANP completely prevented this response. Nuclear run-on analysis showed that increases in intracellular Ca2+ resulted in transcriptional induction of the StAR gene and that ANP inhibited this process. These results demonstrate that Ca2+ exerts a transcriptional control on StAR protein expression and that ANP appears to elicit its inhibitory effect on aldosterone biosynthesis by acting as a negative physiological regulator of StAR gene expression.

Stimulation by interleukin-1 of renal calcium reabsorption in thyroparathyroidectomized rats.

Recombinant human interleukin-1 (rhIL-1) can induce an elevation in calcium that has been ascribed exclusively to the stimulation of bone resorption. In the present study, we investigated whether rhIL-1 could also enhance the renal tubular reabsorption of calcium. The chronic influence of recombinant human rhIL-1 on renal calcium transport was investigated in thyroparathyroidectomized rats. Administration of rhIL-1 at the dose of 1.5 micrograms/day sc for 6 days induced a significant elevation in plasma calcium that was associated with a slight but significant decrease in the urinary excretion of calcium. Recording of the urinary calcium excretion expressed per ml glomerular filtrate at various plasma calcium levels, as achieved by acutely infusing calcium gluconate, indicates that rhIL-1 enhanced the tubular reabsorption of calcium. The calculated index of the tubular reabsorption of calcium (TRCal) was significantly increased by rhIL-1 (2.18 +/- 0.14 versus 1.79 +/- 0.07 mmol/l GFR, p < 0.05, in vehicle-treated rats). The change in the renal handling of calcium was not associated with stimulation of the tubular reabsorption of magnesium. Acute administration of a large dose (24 micrograms given in a bolus IV injection) of rhIL-1 enhanced within minutes the urinary excretion of prostaglandin E2. This effect was followed by a significant increase in urinary cAMP excretion and associated with a lower urinary calcium excretion. In conclusion, the results presented in this study indicate that rhIL-1 administered chronically selectively stimulated the tubular reabsorption of calcium. Experimental evidence suggests that this effect is mediated by prostaglandin-induced cAMP generation. These data strongly suggest that changes in the tubular handling of calcium could contribute to rhIL-1-induced hypercalcemia.

Presence of two neurophysins in the human pineal gland.

The tentative identification of two neurophysins in human pineal glands is reported. The presence of these carrier-proteins for neurohormones was demonstrated by two different radioimmunossays: one highly specific for human pituitary estrogen-stimulated neurophysin (h-ESN) and the second for pituitary bovine neurophysin II (b-NII). These neurophysins accounted for about 0.425% of the total soluble pineal proteins. They were eluted from Sephadex G-75 column with the same elution volume as 125I labeled h-ESN. Their dilution curves were parallel to the standard curves of h-ESN and b-NII, respectively. Electrophoresis on polyacrylamide gel separated two distinct neurophysins. The less anodic neurophysin corresponding to b-NII and the faster moving one to h-ESN. The two proteins were named human pineal neurophysins I and II (h-PNI and h-PNII) in the order of their electrophoretic mobility. Purification of pineal neurophysins by isoelectric focusing resulted also in the separation of two neurophysins: one, recognized by antibody against h-ESN with an isoelectric point (pI) of 4.6, the other recognized by antibody against b-NII with a pI of 4.9. The presence of neurohormone(s), assessed by radioimmunoassay for [8-arginine] vasopressin, was also demonstrated. Under optimal conditions for the association the neurophysin and the neurohormone were eluted from Sephadex columns as a complex. This complex could be dissociated in 0.1N formic acid and the neurophysin and the neurohormone separated on Sephadex G-75.

Plasma vasopressin levels after infusions of hypertonic saline solutions into the renal, portal, carotid, or systemic circulation in conscious dogs.

To determine whether extracerebral osmoreceptors contribute to vasopressin release when exposed to blood osmolality changes of about 1%, we administered hypertonic saline solution to five conscious dogs through catheters chronically implanted into the inferior vena cava, the portal vein, the artery to a sole remaining kidney, and the common carotid arteries. Each infusion was given on a different day at a rate of 0.2 ml/min, which provided about 25 mumol NaCl/kg BW X min. The changes in plasma sodium concentration and plasma osmolality measured during these infusions were similar with all four routes of administration and significantly different from those after iv infusion of isotonic saline solution. Plasma vasopressin concentration, measured by RIA, increased more rapidly after intracarotid infusions than with any other route. We found no evidence that renal or portal/hepatic osmoreceptors contributed to vasopressin release under the conditions of our study.

Abnormal water turnover associated with hypothalamic obesity.

Experimental obesity produced in rats by stereotaxic lesions of the ventromedial hypothalamus (VMH) resulted in hyperphagia, polydipsia, polyuria, decreased urine osmolality, and enhanced excretion of total solute and urea. A 24-h water deprivation test revealed the inability of VMH-lesioned rats to increase urine antidiuretic hormone (ADH) excretion. Thus, destruction of the VMH area appears to be accompanied by impairment of ADH secretion, resulting in a diabetes insipidus syndrome that is partially masked by food restriction and improved by treatment with exogenous ADH.

Stimulation of corticosteroid biosynthesis by angiotensin I [des-asp1]angiotensin I, angiotensin II and [des-asp1]-angiotensin II in bovine adrenal fasciculata cells.

The effect of angiotensin I (AI), angiotensin II (AII), [des-asp1]AI, [des-asp1]AII and [des-asp1-arg2]AII on corticosteroid production in isolated fasciculata cells from bovine adrenals has been studied. AII and [des-asp1]AII in concentrations ranging from 10(-9)M to 10(-6)M had a potent stimulatory effect on steroid biosynthesis. The dose-response curves for both peptides were identical. AI was about 3 times less potent than AII and [des-asp1]AII. The effect of AI was not due to its conversion to AII. [Des-asp1]AI was as active as AI. No significant conversion to [des-asp1]AII was observed. [Des-asp1-arg2]AII had only a minimal effect on steroidogenesis. The structural analog [sar1,-ala8]AII inhibited all angiotensins specifically and competitively. The affinity of the cellular binding site was higher for AII and [des-asp1]AII than for [sar1,ala8]ALL, but lower for AI and [des-asp1]AI than for the inhibitor. Combination of submaximal doses of AI and AII resulted in an additive effect on steroid production. By contrast, combination of maximal doses of both peptides had the same effect as AII alone. These data demonstrate a potent steroidogenic activity for AII as well as AI, [des-asp1]AI and [des-asp1]AII in bovine adrenal fasciculata cells. A common receptor site for all four peptides is suggested.

Role of the capacitative calcium influx in the activation of steroidogenesis by angiotensin-II in adrenal glomerulosa cells.

Angiotensin-II (AngII)-induced Ca2+ influx in adrenal glomerulosa cells, a signal necessary for the stimulation of steroidogenesis by the hormone, is believed to involve two distinct mechanisms: 1) opening of voltage-operated Ca2+ channels, and 2) activation of a capacitative Ca2+ entry pathway that is dependent on calcium release from intracellular stores. Nicardipine, a dihydropyridine calcium antagonist, has been used to investigate the role of these Ca2+ entry mechanisms in the steroidogenic response to AngII. As demonstrated with the patch-clamp technique, micromolar concentrations of nicardipine completely blocked voltage-operated Ca2+ channel activity of both T- and L-types. This agent similarly inhibited the rise of cytosolic free calcium concentration induced by potassium, but did not significantly affect the response to thapsigargin, an activator of the capacitative pathway. Nicardipine reduced by only 22% the calcium influx stimulated by AngII, and the nicardipine-insensitive part of this response was abolished after exhausting the intracellular Ca2+ stores with thapsigargin. Similarly, aldosterone secretion induced by AngII was only partially inhibited (40%) by nicardipine at concentrations that completely abolished the steroidogenic response to potassium. Thapsigargin by itself was able to stimulate aldosterone production, an action highly potentiated by physiological concentrations of extracellular potassium. These data strongly suggest that the major part of the calcium influx response to AngII, leading to aldosterone formation, involves a capacitative calcium entry pathway activated by the release of calcium from intracellular stores. This mechanism of calcium influx could be responsible for some features of aldosterone response to the hormone, such as its poor sensitivity to dihydropyridines or its potentiation by potassium.

Distinct functions of T- and L-type calcium channels during activation of bovine adrenal glomerulosa cells.

Calcium influx into adrenal glomerulosa cells is a key event during the stimulation of aldosterone secretion by physiological increases in extracellular potassium concentrations. Two types of voltage-operated calcium channels, T- and L-types, are present on bovine glomerulosa cells, but their respective functions are not yet clearly defined. Using the patch-clamp method in the perforated patch configuration combined with microfluorimetry of cytosolic calcium, we demonstrate that L-type channels are exclusively responsible for the sustained elevation of cytosolic calcium observed upon stimulation with extracellular potassium, even at low, physiological concentrations of this agonist. In contrast, aldosterone secretion appears closely related to T-type channel activity. Moreover, when the activity of each channel type is selectively modulated by pharmacological agents, such as dihydropyridines or zonisamide, the cytosolic calcium response can be clearly dissociated from the steroidogenic response. Similarly, modulation of T channel activation by protein kinase C results in a parallel inhibition of aldosterone secretion, without any effect on the levels of cytosolic free calcium. This direct functional link between T-type calcium channel activity and steroidogenesis suggests a model in which calcium entering the cell through these channels bypasses the cytosol to activate intramitochondrial steps of aldosterone biosynthesis.

Peripheral-type benzodiazepines inhibit calcium channels and aldosterone production in adrenal glomerulosa cells.

Angiotensin-II (Ang-II), K+, and ACTH are important stimulators of aldosterone secretion that require Ca2+ influx to be active. However, Ang-II and K+ are linked to the Ca2+ messenger system, while ACTH is coupled to the cAMP pathway. Peripheral-type binding sites for benzodiazepines are particularly abundant in steroidogenic tissues and have been proposed to be involved in the steroidogenic action of ACTH in Y-1 adrenocortical cells. We report here that in adrenal glomerulosa cells, peripheral-type [4'-chlor-diazepam (CDZ), 1-(2-chlorophenyl)N-methyl-N-(1-methylpropyl)3-isoquinolinecarboxamid e (RP 52028), and flunitrazepam], but not a central-type (flumazenil) benzodiazepine reversibly abolished the stimulation of aldosterone output induced by Ang-II or K+, while they had no significant effect on basal aldosterone secretion. This inhibitory effect depended upon drug concentration (IC50 30 microM for CDZ) and affected the potencies of both stimulators, without altering their respective EC50 values. Similar results were obtained when aldosterone production was stimulated with ACTH, forskolin, or (Bu)2cAMP. Aldosterone production from exogenous 25-hydroxycholesterol or progesterone was partially inhibited by CDZ. In glomerulosa cells loaded with a fluorescent Ca2+ probe, benzodiazepines blocked Ca2+ influx triggered by K+ or Ang-II without affecting the release of Ca2+ from intracellular stores induced by Ang-II. T- and L-type Ca2+ channel activities, monitored with the patch-clamp technique, were both inhibited within the same range of concentrations as aldosterone synthesis and Ca2+ influx. These results indicate that in adrenal zona glomerulosa cells, peripheral-type benzodiazepines block Ca2+ influx through voltage-activated channels. The combined action of peripheral-type benzodiazepines on calcium influx and precursor conversion may be responsible for the observed inhibition of Ang-II-, K(+)-, or ACTH-induced aldosterone secretion.

Possible role for mitochondrial calcium in angiotensin II- and potassium-stimulated steroidogenesis in bovine adrenal glomerulosa cells.

In adrenal zona glomerulosa cells, the action of angiotensin II (Ang II) and of potassium (K+) on aldosterone synthesis is mediated by the Ca2+ messenger system. The major part of the steroidogenic pathway takes place inside the mitochondria, and Ca2+ must enter the mitochondrial matrix to stimulate the steroidogenic cascade. To examine how changes in the cytosolic free calcium concentration ([Ca2+]c) induced by Ang II and K+ are relayed into the mitochondrial matrix, we transfected bovine adrenal zona glomerulosa cells in primary culture with a chimeric complementary DNA encoding for the signal presequence targeting human cytochrome c oxidase subunit VIII to the matrix, linked to a complementary DNA coding for the Ca2+-sensitive photoprotein aequorin. Resting mitochondrial free calcium concentration ([Ca2+]m) amounted to 0.41 +/- 0.18 microM (n = 40). Ang II induced a concentration-dependent (EC50 = 11.3 +/- 6.0 nM), biphasic rise of [Ca2+]m. After a large transient initial peak (5.13 +/- 0.89 microM, n = 28), [Ca2+]m decreased to a plateau that remained higher than basal [Ca2+]m for several minutes in the presence of the hormone. By contrast, studies in cells transfected with cytosolic aequorin indicated that the rise of [Ca2+]c triggered by Ang II was confined to 1.34 +/- 0.26 microM (n = 17). In Ca2+-free medium, a reduced peak [Ca2+]m response to Ang II occurred without a secondary plateau. On readdition of extracellular Ca2+, in the presence of the hormone, the resulting Ca2+ influx was accompanied by small rise of [Ca2+]m. The mitochondrial uncoupler, carbonyl cyanide p-(trifluoro-methoxy)phenyl-hydrazone, prevented the Ang II-induced [Ca2+]m rise but not the [Ca2+]c response, thus demonstrating the mitochondrial location of transfected aequorin. In contrast to Ang II, K+ (13 mM) induced a sustained [Ca2+]c response, which was relayed without amplification into the mitochondrial matrix as a plateau of[Ca2+]m. This plateau of[Ca2+]m was suppressed by the addition of the dihydropyridine, nifedipine (200 nM). The inhibitor of the mitochondrial Na+/Ca2+ exchanger, CGP37157, reduced significantly the rate of decrease of [Ca2+]m following the peak induced by Ang II. In cells whose [Ca2+]c was clamped at various levels (0.05-0.860 microM) with ionomycin, a concentration-dependent stimulation of pregnenolone output was induced by Ca2+. Under these conditions, the output of pregnenolone--the early product of steroidogenesis--was markedly potentiated by CGP37157. These results suggest the existence of microdomains of high [Ca2+]c elicited by Ang II in the proximity of mitochondria. Moreover, our observations are consistent with a mitochondrial site of action for calcium in the activation of the steroidogenic cascade.

Angiotensin-II induces changes in the cytosolic sodium concentration in bovine adrenal glomerulosa cells: involvement in the activation of aldosterone biosynthesis.

The homeostasis of cytosolic free calcium ([Ca2+]i) and intracellular free sodium ([Na+]i) are linked in many cell types. We, therefore, studied the effect on [Na+]i of two physiological stimulators of aldosterone synthesis that trigger the calcium messenger system, angiotensin-II (Ang II) and potassium ion (K+), in cultured bovine adrenal glomerulosa cells, using the intracellular fluorescent probe for sodium, sodium benzofuran isophthalate. Ang II induced a concentration-dependent and sustained increase in [Na+]i, from a resting value of 9.2 +/- 3.5 to a maximum of 48.5 +/- 5.5 mM (n = 14). This [Na+]i response was mediated by receptors of the AT1 subtype, because it was abolished by losartan (DuP 753). K+ (15 mM) induced a weaker [Na+]i response, from 5.9 +/- 2.6 to 16.8 +/- 2.5 mM (n = 9). In freshly prepared cells, basal [Na+]i was significantly higher (23.9 +/- 1.8 mM; n = 14; P < 0.01) than in cultured cells. Atrial natriuretic peptide, which is known to affect sodium transport in various cell types, did not alter the [Na+]i response elicited by Ang II. Ethylisopropylamiloride, an inhibitor of Na+/H+ exchange, and dichlorobenzamyl, an inhibitor of Na+/Ca2+ exchange, both inhibited in a concentration-dependent manner the Ang II- and K(+)-induced aldosterone response. Isoosmotic replacement of extracellular Na+ markedly reduced basal aldosterone synthesis. Under these conditions, the concentration-response curve for Ang II-induced aldosterone synthesis was shifted to the right, and its maximum was strikingly diminished. These results show that Ang II and, to a lesser extent, K+ induce significant changes in [Na+]i in bovine glomerulosa cells. These [Na+]i changes probably occur through the Na+/H+ and Na+/Ca2+ exchangers and are likely to play a role in activation of the steroidogenic cascade.

Blocking T-type calcium channels with tetrandrine inhibits steroidogenesis in bovine adrenal glomerulosa cells.

Tetrandrine, an alkaloid extracted from a Chinese medicinal herb traditionally used in hypertension treatment, inhibited aldosterone production induced in bovine adrenal glomerulosa cells by either potassium ion, angiotensin II, or ACTH in a concentration-dependent manner (IC50 = 10 microM). The inhibition of the response to potassium by tetrandrine had a pattern very similar to that of nickel, a blocker of T-type calcium channels. In addition, tetrandrine prevented calcium influx induced by potassium or angiotensin II without affecting the calcium release phase stimulated by the hormone. The effect of tetrandrine on voltage-activated barium currents was investigated using the whole cell configuration of the patch clamp technique. T-type currents were isolated by recording the slowly deactivating currents elicited during repolarization of the cell to -65 mV after various depolarizing pulses. These currents were blocked by micromolar concentrations of the drug. The voltage sensitivity of channel activation was not affected by tetrandrine; nevertheless, the drug significantly slowed the deactivation of the current. The action of tetrandrine did not require the activation of the channel. Tetrandrine also affected L-type currents, as assessed after inactivating T channels for 100 msec, but at higher concentrations of the drug. Thus, tetrandrine affects with a similar potency aldosterone production, calcium influx, and T-type calcium channel activity. This finding strongly suggests a role for these channels in calcium signaling and control of steroidogenesis in adrenal glomerulosa cells.

Arginine-vasopressin in postpartum panhypopituitarism: urinary excretion and kidney response to osmolar load.

The response to an osmolar load (750 ml 2.5% NaCl solution iv preceded by 500 ml water by mouth) was studied in 20 patients with Sheehan's syndrome and 12 normal women. Sodium and osmolality were determined in plasma and urine and arginine-vasopressin (AVP) was measured by RIA in urine. The test was performed in each patient when untreated (group P), after hydrocortisone replacement alone (group F), and combined hydrocortisone and thyroid hormone replacement (group F+T). After the osmolar loading, maximum urinary osmolality in the patients was lower than in the normal women and remained unaffected by both hydrocortisone alone and hydrocortisone and thyroid hormone. Comparison of the mean hourly urinary volume before and after NaCl infusion demonstrated an increase in group P, a decrease in group C, and no change in groups F and F+T. Although free water clearance became negative in all groups, values in groups P, F, and F+T were constantly above that of group C. None of the patients in groups P and F had a significant rise in urine AVP excretion during or after NaCl infusion. Those in group F+T had a slight AVP response which was less than in normal women. Impaired response of AVP to an osmolar load appears to be a constant feature of Sheehan's syndrome even without overt diabetes insipidus.

The renin-angiotensin system in panhypopituitarism: dynamic studies and therapeutic effects in Sheehan's syndrome.

To gain insight in the influence of the pituitary gland on the renin-angiotensin system plasma renin substrate (PRS) and the response of PRA to stimulation were studied in a homogeneous group of 20 female patients with the same etiology and degree of pituitary failure, before treatment (group P), after hydrocortisone substitution (group F), and after hydrocortisone and thyroid hormone treatment (group F + T). All patients were studied before and after each treatment by response to two stimulatory tests, acting through two different pathways; orthostasis test (O-T) and the furosemide test (Furo-T). Results were compared between groups, each patient serving as her own control, and with those obtained in a 12 healthy women control group (group C). The diet contained about 85 meq Na/day. Compared to group C (O-T response, 5.97 +/- 0.54 ng ml-1 h-1; Furo-T response, 6.71 +/- 0.82 ng ml-1 h-1; mean +/- SEM), PRA response to both tests was blunted in group P (O-T: 2.48 +/- 0.46, P less than 0.001; Furo-T: 3.02 +/- 0.53, P less than 0.001) and remained so in F (O-T: 2.18 +/- 0.40, P less than 0.001; Furo-T: 2.52 +/- 0.28, P less than 0.001), In group F + T, the response to both tests was greater than in P and F (O-T, 6.61 +/- 1.19; Furo-T, 4.36 +/- 0.44; 0.001 less than P less than 0.05). However, whereas the response to orthostasis is entirely normalized, the response to a diuretic remained significantly smaller than in group C (P less than 0.01). These improvements were observed without significant change in PRS concentration which remained low. We conclude that panhypopituitarism is accompanied by an altered renin angiotensin system. Basal levels of PRS and PRA are low and unresponsive to adequate stimulation. Whereas glucocorticoid therapy alone is without effect on this hyporeninism, addition of thyroid hormones completely normalized the response to orthostasis and significantly improved furosemide response.

Detection of human anti-thyroxine and anti-triiodothyronine antibodies in different thyroid conditions.

Anti-thyroxine (T4) and anti-triiodothyronine (T3) antibodies have been demonstrated in man. It was assumed that antibodies were at least partially saturated in vivo by the hormones. The initial step of the method therefore consisted in a dissociation of the postulated antigen-antibody complex by a 45% ammonium sulfate precipitation. The second part of the method consisted in incubating the euglobulins with trace amounts of 125I-T3 and 131I-T4. The hormones bound to the gammaglobulins were then separated from the free hormones by a column of DEAE Sephadex A-50 in ammonium acetate 0.05 M pH 7.6. The amounts of 125I and 131I bound to the gammaglobulin fraction were then measured. The results in any unknown sample were compared to those obtained when an equal amount of standard serum was identically treated, and the results were expressed as unknown/standard ratios of bound 125I and bound 131I, respectively. The mean binding ratios for T3 and T4 found in sera obtained from 42 normal subjects were 0.7 +/- 0.4 SD and 0.8 +/- 0.5, respectively. Elevated binding ratios for both T3 and T4 were found in sera obtained from 5 out of 43 cases of primary hypothyroidism and in 2 out of 34 cases of hyperthyroidism. The binding ratios were elevated for only T3 in 10 cases of primary hypothyroidism and in 5 cases of hyperthyroidism. Antibodies against T4 were detected in one case of primary hypothyroidism. High binding ratios for T3 were also observed in one patient with secondary hypothyroidism who had received treatment with dessicated thyroid for several years. In most of the positive sera, anti-thyroglobulin antibodies, as measured by passive hemagglutination, could also be detected. For one serum containing anti-T3 antibody and another containing anti-T4 antibody, the binding affinity and capacity were estimated by Scatchard plot analysis; affinity constants were 5.4 x 10(8) L/mol and 1.3 x 10(9) L-mol, respectively; capacities 1.4 ng/ml and 1.2 ng/ml, respectively. The presence of anti-T3 and anti-T4 antibodies in serum may result in an apparent lowering of the serum T3 and T4 concentrations, respectively.

Radioimmunoassay for 3,3',5'-triiodo-L-thyronine in unextracted serum: method and clinical results.

Serum 3,3',5'-triiodothyronine (rT3) was measured with a radioimmunoassay in unextracted serum. The assay was specific and reproducible. The coefficients of variation for 3 different sera known for high, normal, and low rT3 concentrations between assays were 4, 6, and 9% within assays 4, 9, and 7%, respectively. In euthyroid subjects 20 to 60 years old, rT3 was 450 +/- 200 pg/ml (mean +/- 2 SD.n = 83). Serum rT3 was found to be increased in hyperthyroidism (range: 762-2581 pg-ml; n = 11) but also in acute and chronic illness (up to 2400 pg/ml; n = 24) and in anorexia nervosa (536-1058 pg/ml; n = 7). In the latter two situations there was mostly an inverse change in serum 3,5,3'-triiodothyronine (T3) which was in the low normal range or decreased. These findings suggest a metabolic control of thyroxine deiodination. A low serum rT3 was found in 9 of 12 hypothyroid patients and in the serum of 1 chronically ill patient. Long-term treatment (1-7 years) with lithium carbonate slightly reduced serum rT3, although the changes were inside the normal range. Kidney function was not found to be necessary for its production as anephric patients had normal rT3 values. In addition, hemodialysis increased serum rT3, which is probably due to the heparin therapy.

A novel point mutation in the translation initiation codon of the pre-pro-vasopressin-neurophysin II gene: cosegregation with morphological abnormalities and clinical symptoms in autosomal dominant neurohypophyseal diabetes insipidus.

Autosomal dominant neurohypophyseal diabetes insipidus (ADNDI) is a rare variant of idiopathic central diabetes insipidus. Several different mutations in the human vasopressin-neurophysin II (AVP-NP II) gene have been described. We studied nine family members from three generations of an ADNDI pedigree at the clinical, morphological, and molecular levels. AVP concentrations were measured during diagnostic fluid restriction tests. Coronal and sagittal high resolution T1-weighted images of the pituitary were obtained from affected and healthy family members. PCR was used to amplify the AVP-NP II precursor gene, and PCR products were directly sequenced. Under maximal osmotic stimulation, AVP serum levels were close to or below the detection limit in affected individuals. Magnetic resonance imaging studies revealed the characteristic hyperintense ("bright spot") appearance of the posterior pituitary in two healthy family members. This signal was absent in all four ADNDI patients examined. The coding sequences of AVP and its carrier protein, neurophysin II, were normal in all family members examined. Affected individuals showed a novel single base deletion (G 227) in the translation initiation codon of the AVP-NP II signal peptide on one allele. The mutation in the AVP-NP II leader sequence appears to be responsible for the disease in this kindred, possibly by interfering with protein translocation. The absence of the hyperintense posterior pituitary signal in affected individuals could reflect deficient posterior pituitary function.

Potentiation of the antagonistic effect of ACTH11-24 on steroidogenesis by synthesis of covalent dimeric conjugates.

Covalent dimerization of the adrenocorticotropin fragment ACTH11-24 increases its antagonistic potency on the ACTH-induced steroidogenesis in isolated bovine fasciculata/reticularis cells by 3 orders of magnitude when the C-termini are linked via a 10 A long spacer. This strong potentiation, probably mediated by cross-linking of the receptors, was shown to be dependent on the point of attachment of the monomeric fragment to the spacer, thus providing information about the position of the binding site in the hormonal segment and about the distance of the receptors on the cell surface.