Afrikander cattle congenital goiter: characteristics of its morphology and iodoprotein pattern. The morphology and some properties of the complex iodoprotein pattern of the genetically determined congenital goiter in Afrikander cattle is described. The goiter contained irregularly shaped follicles which were devoid of colloid and the follicular epithelial cells were elongated, measuring about 20 micrometer in length compared to 10 micrometer for normal thyroid cells. The goiter cells contained apical clusters of larger and more numerous lysosomes than normal thyroid cells. Apical vesicles containing electron-dense material which were in contact with the plasma membrane could be seen in most normal thyroid cells, but were extremely scarce in the goiter. In 36 cell profiles studied none was found. The endoplasmic reticulum cisternae of the goiter differed significantly from normal thyroid cells. Fewer ribosomes were seen to be attached to the membranes of goiter cells. Furthermore, unlike normal thyroid cells, many free polysomes were seen in goiter cells. The characteristics of the unusual iodoprotein pattern of the goiter extract, resolved by gel chromatography and sucrose density gradient centrifugation, were qualitatively and quantitatively similar to that described previously (Endocrinology 91, 470, 1972). A relatively small amount of the total soluble protein was iodinated. Of these, only a 12S sedimenting species was precipitated by antithyroglobulin immunoglobulin. When separated on polyacrylamide gels containing sodium dodecyl sulfate and mercaptoethanol, this 12S species was resolved into at least 14 polypeptide components ranging in molecular weights from less than 66,000--330,000. Three of the bands, representing a small percentage of the total protein, seemed to comigrate with the major polypeptides of thyroglobulin and were also precipitated with rabbit antihyroglobulin immunoglobulin. The data indicate that glycosylation of iodoproteins was not affected although 19S thyroglobulin synthesis and subsequent storage were drastically impaired.


Reduction of granulosa cell progesterone secretion in vitro by intraovarian implants of antiandrogen. Intra-ovarian Silastic implants containing the antiandrogen, Flutamide, or its active metabolite, reduced the level of progesterone secretion in porcine granulosa cell cultures by approximately 50%. These results complement recent observations in vitro suggesting a role for androgen in granulosa cell steroidogenesis during follicular development.


Participation of serotonin in the phasic release of luteinizing hormone. II. Effects of lesions of serotonin-containing pathways in the central nervous system. Circadian LH variations were measured in castrated, estradiol-implanted female rats bearing lesions of the raphe nuclei, in which most serotonin (5-HT) containing ascending projections originate, or mediopontine transections interrupting such projections before they enter the hypothalamus. Previous data indicated that pharmacological blockade of 5-HT biosynthesis abolishes the rhythmic pattern of LH secretion; the present study was intended to check whether surgical depletion of hypothalamic 5-HT had a similar effect and whether such abolition could be correlated with hypothalamic or forebrain endogenous concentrations of the amine and of its metabolite, 5-hydroxyindoleacetic acid (5-HIAA). Maximal inhibition of hypothalamic 5-HT and 5-HIAA concentrations were obtained after complete basal mediopontine transections and after lesions of the medial and the dorsal raphe nuclei; under these conditions, the daily variations in plasma LH were reduced by more than 70%. Smaller lesions, even when they completely destroyed the medial raphe, were much less effective. A good correlation was observed between the amplitude of the LH cycle and the extent of hypothalamic, but not of forebrain, 5-HT and 5-HIAA depletion. However, and in contrast to the results of pharmacological 5-HT inhibition, the cyclic pattern of LH secretion could not be totally abolished by any of these surgical procedures. It is concluded that the dorsal raphe contributes to the regulation of rhythmic LH secretion in castrated female rats bearing estradiol implants by modulating the amplitude of this circadian cycle rather than by generating the rhythmic pattern itself.


The effect of blockade of dopamine receptors on the inhibition of episodic luteinizing hormone release during electrical stimulation of the arcuate nucleus in ovariectomized rats. This study examined the possible involvement of dopamine (DA) in mediating the inhibition of episodic LH release that occurs during electrical stimulation of the arcuate nucleus (ARH) in ovariectomized rats. Animals were treated before stimulation with pimozide (1.26--2.0 mg/kg) or d-butaclamol (1 mg/kg), blockers of DA receptors, or l-butaclamol. Apomorphine, which inhibits episodic LH release by activating DA receptors, was given near the end of the experiment to determine if these receptors were blocked. ARH stimulation suppressed pulsatile LH release in six rats when DA receptors were not blocked by pimozide (as well as two in which blockade was not tested). A transient increase occurred in one other animal. When DA receptors were blocked by pimozide, stimulation of the ARH inhibited episodic LH release in nine rats, suggesting that DA may have no role in mediating this inhibition. However, because increased LH release occurred in five additional animals, as well as in one with partial receptor blockade, the possibility remains that DA may perhaps have a minor role in this inhibitory response.  Although ARH stimulation increased LH release after DA receptor blockade by d-butaclamol, this effect could not be ascribed to the DA antagonist property of this agent, because elevated blood LH levels also occurred during stimulation in rats treated with l-butaclamol, in which DA receptors were not blocked. d- and l-butaclamol may possess a non-stereospecific action on a non-dopaminergic event, thus reversing the response to ARH stimulation. Finally, whether DA receptors were blocked or not by pimozide, d-, or l-butaclamol, activation of the ventromedial hypothalamic and periventricular nucleus regions suppressed episodic LH release, but did not increase LH secretion. This suggests that the region through which stimulation can inhibit, but not increase, LH release may extend in the hypothalamus to these two areas.


Sigmoidal relationship between parathyroid hormone secretion rate and plasma calcium concentration in calves. Parathyroid hormone (PTH) secretion rate was measured in 16 anesthetized calves by using a technique involving radioimmunoassay of parathyroid venous blood which was collected during timed intervals and measured volumetrically. The calves ranged in age from 2-14 weeks. Plasma calcium concentration was altered by infusion of solutions of CaCl2 or disodium ethylenediamine tetracetate (Na2 EDTA) into the jugular vein. When plasma calcium concentrations exceeded 10.5 mg/100 ml, a basal, non-suppressible secretion rate of 0.3 ng/kg/min was maintained despite the induction of hypercalcemia. Slight changes in secretion rate were observed in response to changes of plasma calcium in the range between 9 and 10.5 mg/100 ml. Below 9 mg/100 ml, a small decrease in plasma calcium concentration evoked a pronounced increase in secretion rate. A maximal secretion rate of about 5.5 ng/kg/min was attained at a plasma calcium concentration of approximately 7.5 mg/100 ml and it was not increased by more severe hypocalcemia. These observations confirm the sigmoidal relationship between PTH secretion rate and  plasma calcium concentration which was previously suggested by measurement of PTH concentration in peripheral plasma of hypocalcemic, parturient cows.


Further studies on the relationship between potassium and sodium levels and adrenocortical activity. Steroid production in vitro by dog adrenal cortical slices was measured in the presence of varying concentrations of sodium and potassium ions. Increasing concentrations of potassium produced a significant increase in the intracellular potassium content and in the rate of synthesis of aldosterone. However, the potassium effect on aldosterone secretion may also occur without changes in tissue potassium content. Ouabain significantly diminished intracellular potassium content, but inhibited aldosterone production only at the high dose of 2.5 X 10(-3) M in the presence of an elevated external potassium level. Physiological changes in sodium concentration can modify aldosterone production. The effect was observed with changes as small as 10 mEq/liter sodium. Changes in external potassium and sodium levels modulate aldosterone as well as corticosterone, but not cortisol production. No changes in the intracellular content of potassium were detectable in angiotensin-stimulated tissue. Nevertheless, high doses of ouabain blocked angiotensin-stimulated aldosterone production without affecting cortisol production. The dissociation observed between intracellular potassium levels and aldosterone production suggests that there is not a simple relationship  between the two parameters.


Effect of testosterone and progesterone on the estradiol receptor in the immature rat ovary. The effect of testosterone and progesterone on the estrogen-stimulated ovarian weight increase and es tradiol receptor (ER) levels was examined in immature, hypophysectomized rats. Nuclear translocation and cytosol replenishment of the ER in the ovary exhibited similarities to those events in the uterus. The ovarian weight increase observed on day 4 in response to three daily injections of 2.5 microgram 17beta-estradiol administered sc in sesame oil was effectively suppressed by one injection of 1 mg testosterone on day 3. In contrast, 1 mg progesterone given on day 3 had no effect on the ovarian weight of the estrogen-primed rat. Nor did progesterone alter the ovarian ER levels in the cytosol and nuclei of similarly treated rats. Further, progesterone receptors were undetectable in the ovarian tissue of immature, hypophysectomized rats given two daily injections of 2.5 microgram estradiol-17beta. In contrast, testosterone significantly reduced the ovarian ER levels both in the cytosol as well as in the nuclei. These results suggest that one of the mechanisms by which testosterone acts in the ovarian tissue is by reducing the available ER for estrogen action.


The binding of 5 alpha-pregnane-3,20-dione by cytosol and nuclear preparations of guinea pig uterus. We compared the binding characteristics of [3H]progesterone and 5 alpha-[3H]pregnanedione in cytosol and nuclear preparations of ovariectomized, estrogen-treated guinea pig uterus. There were similarities as well as differences in binding behavior. [3H]Progesterone and 5 alpha-[3H]pregnanedione were bound in cytosol with approximately the same apparent association constants and concentrations of binding sites. When centrifuged on sucrose gradients in 5 mM phosphate buffer, binding peaks with sedimentation coefficients of 7S were found with both. 5 alpha-[3H]Pregnanedione was bound by uterine nuclei and, like [3H]progesterone, required temperature-activated cytosol of estrogen-stimulated uterus. A series of unlabeled steroids had similar relative abilities to displace both [3H]progesterone and 5 alpha-[3H]pregnanedione from receptor complexes in cytosol or nuclei. When cytosol was incubated with increasing concentrations of [3H]progesterone or 5 alpha-[3H]pregnanedione the amount of 5 alpha-[3H]pregnanedione bound exceeded the amount of [3H]progesterone bound at steroid concentrations above 5 x 10(-9) M. This suggested that 5 alpha-pregnanedione was bound by additional components of the cytosol and the suggestion was strengthened by sucrose gradient analysis. At greater than saturating-steroid concentrations, the partition between 7S and 4 S binding proteins was different. [3H]Progesterone bound to 7S binding proteins in preference to 4S binders, whereas 5 alpha-pregnanedione showed relatively more 4S binding.


Monodeiodination of 3,5,3'-triiodothyronine and 3,3',5'-triiodothyronine to 3,3'-diiodothyronine in vitro. To study conversion of 3,5,3'-triiodothyroinine (T3) and 3,3',5'-triiodothyronine (rT3) to 3,3'-diiodothyronine (T2) in vitro, T3 or rT3 was incubated at pH 7.35 with homogenates of several rat tissues (liver, kidney, muscle, heart, ling, spleen, intestines, and brain) for 15 min at 37 C. The T2 generated during incubation was measured in an ethanol extract of the incubation mixture by a specific RIA of T2; T4, T3, and rT3 cross-reacted in the T2 RIA only to an extent of 0.006, 0.2, and 0.04%, respectively. T2 was produced regularly when T3 or rT3 was incubated with liver or kidney homogenates; other tissues generated little or no T2 under similar conditions. Studies with liver homogenates revealed that production of T2 from both T3 and rT3 was influenced significantly by tissue and substrate concentractions, temperature, pH and duration of incubation. T3- as well as  rT3-monodeiodinating activities were unaffected by large doses (greater than or equal to 3 micrometer) of sodium iodide, diiodotyrosine, and methimazole, but were inhibited in a dose-dependent manner by propylthiouracil, iodiacetic acid, and dinitrophenol. The apparent Km for conversion of T3 to T2 approximated 6.0 micrometer and that for conversion of rT3 to T2' 65 nM. Propylthiouracil and iodoacetic acid inhibited conversion of both T3 and rT3 to T2 in an uncompetititve and a non-competitive manner, respectively. The various data suggest that 1) monodeiodination of T3 and rT3 to T2 is enzymic in nature; 2) liver and kidney may be the major sites of metabolic transformations of T3 and rT3 to T2.


Suppression of liver cell proliferation by glucocorticoid hormone: a comparison of normally growing and regenerating tissue in the immature rat. The influence of glucocorticoid hormone on the time-course of liver regeneration in the immature rat has been studied by direct measurement of the rate of DNA accretion after the stimulus of partial hepatectomy. In contrast to hepatocyte proliferation associated with normal growth, which almost completely abolished by small doses of glucocorticoid, it is shown that even enormous amounts of hormone produce, at most, about a halving of the intrinsic cell proliferation rate in regenerating liver. Although deceptively magnified by the exponential growth pattern of the hepatic remnant, the inhibition of cell proliferation is thus considerably less complete than that induced in normally growing liver by much lower doses of hormone, a finding at distinct variance with the conclusions of earlier studies based entirely upon observations of radioactive precursor incorporation rather than direct measurement of DNA accretion. The mechanism by which a regenerative stimulus causes hepatocyte proliferation to lose its normal sensitivity to suppression by glucocorticoid, and thereby to exhibit a steroid insensitivity characteristic of other tissues in which cell proliferation reflects cell replenishment rather than normal growth, remains unknown.


Effect of alloxan-induced diabetes on intestinal peptidases in the rat. Intestinal transport of amino aicds, similar to sugar absorption, is enhanced in experimental diabetes. Because peptidases play a significant role in peptide digestion, we examined the effect of diabetes on intestinal peptidases. Leucyl-naphthylamidase and leucyl-glycine hydrolase (brush border peptidases) and prolyl-glycine hydrolase (cytosol peptidase) were assayed in the brush border and cytosol fraction in diabetic rats 7 days after alloxan administration. Mucosal weight, protein concentration, and total and specific activity of leucyl-naphthylamidase and leucyl-glycine hydrolase were significantly increased in diabetes in the brush border but not in cytosol fraction. By contrast, prolyl-glycine hydrolasw was not affected in cytosol fraction or brush border. These data indicate that brush border peptidases are increased in experimental diabetes. This adaptive response of the small intestinal mucosa is similar to disaccharidase elevation and alteration in the intestinal absorptive function which occurs in experimental diabetes.


Ontogeny of pulsatile luteinizing hormone and testosterone secretion in male lambs. The ontogeny of pulsatile LH and testosterone (T) secretion was studied in the developing male sheep. Three variables of LH secretion (frequency of LH pulses, magnitude of LH pulses, and the levels between pulses) were determined during a 6-h period at each of 1, 2, 3, 5, 7, 8, 12, and 16 weeks of age in six male lambs. Pulsatile LH secretion was evident by 1-2 weeks of age in rapidly growing lambs and by 3-7 weeks of age in slowly growing lambs. Although mean height of LH discharges increased 2-fold after the onset of pulsatile release, levels between discharges remained constant throughout the entire study. The most striking change, however, occurred in the frequency of LH discharges. The frequency increased 20-fold between weeks 1 and 8 and then decreased 3-fold between weeks 8 and 16. In each lamb, a brief (less than 3 h), but marked rise (greater than 2 ng/ml) in levels of circulating T occurred after the first observed pulse of LH. Thereafter, as the lambs became older, increased concentrations of serum T were observed only during periods of pulsatile LH secretion, the highest mean level of T occurring about 1 h after each LH peak. Administration of human chorionic gonadotropin (hCG) iv to 2-day-old males resulted in a marked increase in circulating T. A greater increment in concentrations of serum T occurred after the same absolute dose (50 IU) of hCG at 8 weeks of age than at 2 days of age. These results indicate that in the male lamb 1) pulsatile secretion of LH is initiated shortly after birth and changes in mean levels of LH are primarily a reflection of changes in frequency of LH discharges; 2) before the onset of pulsatile LH secretion, the testes are capable of responding to an exogenous LH-like gonadotropin with increased secretion of T; 3) after the onset of pulsatile LH secretion, a rise in circulating T follows each pulse of LH; and 4) an increase in capacity of the testes to secret T occurs during the first 8 weeks of postnatal life. It is concluded that the pattern of LH secretion, coupled with the capacity of the testes to secrete T, determines the pattern and magnitude of T secretion in the developing male sheep.


Effect of glucagon and insulin on the growth of neonatal rat hepatocytes in primary tissue culture. Commercial (bovine-porcine) glucagon added in a single dose between 10(-12) and 10(-7) M to neonatal rat hepatocytes in primary cultures with subsequent incubation for 20-24 h, stimulated their entry into the DNA synthesis phase as revealed by [3H]thymidine-labeling and radioautography; about 14 h of incubation was required before an effect was observed. Commercial (bovine) insulin at doses between 10(-11) and 10(-7) M apparently stimulated the entry of hepatocytes into S phase. However, insulin's effect, which needed 20 h for induction, was due to the release of a wave of synchronized hepatocytes from an earlier produced block near the G1/S boundary of their growth-division cycle. Equimolar mixtures of glucagon with insulin from 10(-15)-10(-7) M increased the fraction of hepatocytes synthesizing DNA first at 4-8 h, and then at 20-24 h. Effective doses of glucagon, insulin, and glucagon plus insulin also increased the entry of hepatocytes into mitosis, as found after a 4-h incubation with colchicine (0.1 mM). Withholding inactivated fetal bovine serum from the growth medium for 24 h did not change the mitotic activity either of the untreated or of the glucagon- and glucagon plus insulin-stimulated hepatocytes, but it increased the proliferogenic effect of bovine insulin. Highly purified crystalline (porcine) glucagon, insulin, and glucagon plus insulin also stimulated the growth of hepatocytes in the presence or absence of serum. Finally, equimolar (10(-14) M) mixtures of glucagon with (Bu)2cGMP and of insulin with (Bu)2cAMP increased the hepatocytic replication as efficiently as did glucagon plus insulin at the same dose. The present results show that glucagon and insulin are synergistic, intracycle regulators of the growth of neonatal rat hepatocytes. They also suggest that cyclic necleotides may mediate at least partly the hepatotropic effects of the pancreatic hormones.


Role of intraluteal estrogen in the regulation of the rat corpus luteum during pregnancy. The pronounced aromatizing ability of the rat corpus luteum and the ability of estradiol to maintain luteal function. To examine this hypothesis, rats were treated with either estradiol (100 microgram/day), high or low levels of testosterone via Silastic capsules (20 cm or 1 cm in length), or dihydrotestosterone (20-cm capsule) after hypophysectomy and hysterectomy on day 12 of pregnancy. Hypophysectomy and hysterectomy caused serum progesterone and androgen levels, estradiol concentrations in the corpora lutea, and the content of estradiol receptor in luteal cell nuclei to decrease significantly, and caused the cessation of luteal growth. The daily administration of 100 microgram estradiol or of high levels of testosterone via the 20-cm Silastic capsule increased the estradiol concentration in the corpora lutea dramatically, maintained serum progesterone from day 12 through day 15 at concentrations similar to those in pregnant, sham-operated animals, and increased the nuclear content of estradiol receptor in the corpora lutea. Treatment with the small testosterone capsule maintained the serum androgen and progesterone levels, estradiol concentrations in the corpora lutea, and luteal growth at levels observed in pregnant, sham-operated animals. Treatment with the 20-cm dihydrotestosterone capsule did not sustain progesterone secretion and luteal growth. These results suggest that estrogen formed by aromatization within the corpora lutea may play an important role in the regulation of luteal function during pregnancy in the rat.


A cytoplasmic progesterone receptor in hen pituitary and hypothalamic tissues. The cytosol fraction of the anterior lobe of the pituitary in laying White Leghorn hens was shown to possess a progesterone-binding component which perferentially bound progesterone. A similar component was also found in the cytosol of the hypothalamus and of the oviduct magnum. The dissociation constant (Kd) at equilibrium was determined as 1.68 +/- 0.10 (SE) X 10(-10) M in the pituitary, 3.29 +/- 0.10 x 10(-10) M in the hypothalamus, and 3.02 +/- 0.40 x 10(-10) M in the oviduct magnum, whereas the number of binding sites/mg cytosol protein was 5.99 +/- 0.084 (SE) X 10(-13) mol in the pituitary, 0.43 +/- 0.003 x 10(-13) mol in the hypothalamus, and 2.88 +/- 0.114 x 10(-13) mol in the oviduct magnum. The results suggest that a cytoplasmic progesterone receptor is present in the pituitary and hypothalamic tissue, as well as in the oviduct magnum, of the hen.


Hereditary congenital goiter with thyroglobulin deficiency in a breed of goats. Hereditary congenital goiter occurring in an inbred breed of goats is reported. The goitrous goats were severely hypothyroid. Thyroglobulin-related antigens could only be detected by radioimmunoassay in amounts of 0.03% of the total protein concentration in the 105,000 x g supernate of the goitrous gland, which corresponded with 8 microgram soluble thyroglobulin-related antigens/g tissue. After treatment with digitonin, 4 microgram thyroglobulin-related antigens/g tissue could be extracted from the sediment. This means that the gland contained about 12 microgram thyroglobulin-related antigens/g tissue (normal about 100 mg/g). However, no thyroglobulin (19S) could be detected by ultracentrifugation experiments, immunodiffusion, or immunoelectrophoresis. Iodinated proteins with sedimentation constants of 3-9S were found. Among these iodinated macromolecular compounds, iodinated "albumin" and some non-hydrolysable iodinated material was found. These last mentioned components were also found in the blood, indicating a leakage from the gland. As described for comparable cases, these results indicate a defect in thyroglobulin synthesis or its regulation.


Changes of vascular reactivity induced by low vasopressin concentrations: interactions with cortisol and lithium and possible involvement of prostaglandins. Arginine vasopressin in physiological concentrations potentiated the vascular effects of various vasoconstrictor agents. By using the isolated rat mesenteric artery preparation, the pressor effects of norepinephrine, angiotensin II, and potassium chloride were all significantly increased when vasopressin was added to the perfusion buffer. Cortisol and lithium both inhibited the potentiating effect of vasopressin but had no effect on the control pressor response to norepinephrine. When the vascular effects of norepinephrine were first blocked with indomethacin and then restored by the addition of prostaglandin E2, the potentiation by vasopressin was almost completely prevented. This suggests that vasopressin may be acting by stimulating prostaglandin biosynthesis. Cortisol and lithium may exert their inhibitory effects by preventing the activation of prostaglandin synthesis by vasopressin. These findings may be of clinical significance because the phenomena occur well within the range of vasopressin levels found in human plasma.


Vasopressin-dependent adenylate cyclase activities in the rat kidney medulla: evidence for two separate sites of action. This study demostrates the existence of an adenylate cyclase sensitive to vasopressin in the medullary portion of the rat thick ascending limb. Maximal adenylate cyclase stimulations achieved in that segment (31-fold) were higher than those obtained in collecting tubules from the same rats (22-fold). From comparisons of absolute maximal responses it can be calculated that thick ascending limbs account for about 80% of the response to vasopressin of a kidney medulla homogenate. The apparent Km value of adenylate cyclase activation (from 10(-9)-2 x 10(-8) M) in thick ascending limbs was higher in each experiment than that simultaneously measured in the collecting tubules from the same rats (2 x 10(-10)-3 x 10(-9) M). Such a lower sensitivity is probably not due to a greater hormone degradation by the thick ascending limb samples. Experiments using structural analogues of the oxytocin series ([deamino-6-carba]oxytocin and vasotocin) did not give evidence for different vasopressin receptors in the thick ascending limb and the collecting tubule. A step beyond the hormone-receptor interaction, thus, must account for the different patterns of adenylate cyclase response to vasopressin of these two segments.


Vitamin D and cartilage. I. In vitro metabolism of 25-hydroxycholecalciferol by cartilage. In the present work, the capacity of cartilage to metabolize 25-hydroxycholecalciferol was investigated. Cartilage preparations from growth plate, articular surface, rib, scapula, and ear were isolated from 3-week-old normal rabbits and chickens. Each tissue was separately incubated with tritiated 25-hydroxycholecalciferol (, x 10(-9) M) for 1-24 h. Incubations of kidney and muscle were performed simultaneously for comparison. Similarly, cultured chondrocytes isolated from rabbit growth plate and articular cartilage were incubated for 1 or 20 h in medium free of fetal calf serum. After methanol-chloroform extraction of tissues, cells, and their respective media, chloroform phases were chromatographed on Sephadex LH-20 columns. The results show that kidney and cartilage are able to convert 25-hydroxycholecalciferol into a derivative which migrates in the 24,25-dihydroxycholecalciferol region. Cartilage tissue previously boiled is unable to metabolize 25-hydroxycholecalciferol. The conversion of 25-hydroxycholecalciferol occurs with all types of cartilage and is also observed in incubations of cultured chondrocytes. In the latter, the polar 25-hydroxycholecalciferol derivative is detected as early as 1 h after addition of 25-hydroxycholecalciferol. Two findings suggest that the polar derivative of 25-hydroxycholecalciferol produced by cartilage is 24,25-dihydroxycholecalciferol: 1) the cartilage derivative and 24,25-dihydroxycholecalciferol (synthetic and biosynthetic) comigrate during Sephadex LH-20 and high liquid pressure chromatography; and 2) both the cartilage derivative and 24,25-dihydroxycholecalciferol are sensitive to periodate treatment.


Localization of alpha-melanocyte-stimulating hormone in rat brain and pituitary. The distribution of alpha-melanocyte-stimulating hormone (alpha-MSH) was studied in the rat brain with an immunoperoxidase technique. alpha-MSH-containing cells were found in the arcuate nucleus of the hypothalamus. Cells staining for alpha-MSH were also localized in the intermediate lobe of the pituitary. alpha-MSH-containing nerve fibers extended throughout regions of the hypothalamus, thalamus, and midbrain. Two weeks after hypophysectomy, alpha-MSH-positive cells anf fibers were still present in the brain. These results indicate that alpha-MSH of non-pituitary origin is synthesized and stored by neural structures in the rat brain. The detection of alpha-MSH by radioimmunoassay in the rat brain and pituitary supports these observations.


Specific binding of prostaglandins E2 and F2alpha by membrane preparations from rat skin. Specific binding of [3H]prostaglandin E2 ([3H]PGE2) and [3H]prostaglandin F2 alpha ([3H]PGF2 alpha) to plasma membrane and smooth endoplasmic reticulum fractions prepared from rat skin was demonstrated by the Millipore filter assay system. Specific binding was greater in the smooth endoplasmic reticulum fraction. Maximum binding was attained in the presence of Ca++ (0.5 x 10(-3) M), pH 7.2, and at a temperature of 37 C. Unlabeled PGE2 at a concentration of approximately 700 x 10(-9) M inhibited binding to the smooth endoplasmic reticulum fraction by approximately 90%. Other unlabeled prostaglandins, PGE1, PGF2 alpha, and a PGE2 analog, 7-0-13-prostynoic acid, at the same concentration inhibited binding by 40%, 25%, and 30%, respectively. A variety of unlabeled fatty acids (palmitic, oleic, linoleic, eicosatrienoic, and eicosatetraenoic acids), at the higher concentration of approximately 1700 x 10(-9) M, inhibited binding less than 30%, suggesting the presence of specific receptors for PGE2 in the smooth endoplasmic reticulum. Similar results were obtained for the competitive binding studies with [3H]PGF2 alpha. Proteolytic digestion of the membrane fraction by trypsin and pronase, or boiling for 15 min caused marked inhibition of binding, suggesting that the receptors for PGE2 and PGF2 alpha have a protein component. The Scatchard plot analysis of the equilibrium-binding data of [3H]PGE2 and [3H]PGF2 alpha to normal smooth endoplasmic reticulum fractions revealed an apparent dissociation constant (Kd) of 1.1 x 10(-9) M with a binding site concentration of 75 x 10(-12) M for [3H]PGE2, and a Kd of 1,0 x 10(-9) M with a binding site concentration of 35 x 10(-12) M for [3H]PGF2 alpha. These data indicate a greater concentration of binding sites for PGE2 than PGF2 alpha in the normal skin smooth endoplasmic reticulum. On the other hand, analysis of smooth endoplasmic reticulum from skin of essential fatty acid-deficient rats revealed a Kd of 1.2 x 10(-9) M with a binding site concentration of 75 x 10(-12) M for [3H]PGE2, and a Kd of 2.2 x 10(-9) M with a binding site concentration of 175 x 10(-12) M for [3H]PGF2 alpha. The concentration of binding sites for PGF2 alpha in the smooth endoplasmic reticulum of the skin of these rats is increased 5-fold when compared to normal values, whereas that of PGE2 was not altered. These results suggest a possible alteration of PGF2 alpha specific binding to skin endoplasmic reticulum during the pathophysiological abnormalities that accompany essential fatty acid-deficiency syndrome.


Maintenance of serum somatomedin activity in hypophysectomized pregnant rats. Pregnant female rats were hypophysectomized on the 14th day of pregnancy without fetal loss or premature delivery. Completeness of hypophysectomy was confirmed by measurements of serum growth hormone and prolactin. Serum somatomedin activities were measured by the hypophysectomized rat costal cartilage in vitro assay using a human serum standard. In this assay somatomedin activity of normal rat serum is about four times that of normal human serum. Forty-eight hours after hypophysectomy serum somatomedin was maintained at 3.9 +/- 1.0 units/ml. At this time after operation somatomedin activity of nonpregnant hypophysectomized rat serum would be virtually undetectable. Somatomedin concentrations were still maintained on the 18th and 20th day of pregnancy. However, within 24 hours after delivery somatomedin had fallen to less than 0.6 units/ml and by three days post delivery were less than 0.2 units/ml. These observations indicate that the fetal-placental unit, probably by secreting a chorionic somatomammotropin, can maintain the concentration of growth hormone-dependent somatomedin in tha absence of the pituitary.


Decreased thyroid function and high plasma prolactin levels in rats of the Buffalo strain. Rats of the inbred Buffalo strain have previously been reported to be susceptible to thyroiditis, as defined by histology. We have studied the endocrinology of the pituitary-thyroid axis of this strain by making direct measurements of the plasma concentrations of TSH and T4 in untreated, adult Buffalo rats of both sexes.  Plasma PRL levels were also measured. All hormone determinations were by RIA. In addition, relative thyroid weights were noted and, in many cases, preliminary assessment of thyroid histology was made. Our principle findings were as follows. 1) Decreased thyroid function, in addition to the previously reported histological abnormalities, was found to occur spontaneously among the rats studied. Indications of decreased function included elevations of plasma TSH and thyroid weight and depressions of plasma T4. We estimated the incidence of unequivocal thyroid disease as approximately 3% in each sex. 2) Basal plasma PRL concentrations of Buffalo rats averaged three to four times higher than those of outbred CD rats. Our findings strongly suggest that rats of the Buffalo strain will provide a good model for the study of thyroid failure of varying degrees and concomitant changes in the circulating levels of pituitary hormones.


Accumulation and localization of radiocalcium in the rat thyroid gland. To determine the rate of accumulation of radiocalcium (45Ca) in the thyroid gland, the concentration of radiocalcium was measured in the blood serum and thyroid gland at various time intervals up to 7 days after a single ip injection of 45Ca in rats. The ratio of the 45Ca concentration in thyroid gland to serum (T:S) increased with the time interval after injection. Thyroid activity was varied by hypophysectomy or by feeding a low iodine diet with or without added thiouracil. The more active the gland, the faster it equilibrated with the serum 45Ca and the less was the T:S in the steady state. The equilibration was much slower than for radioiodide, taking several days in normal control rats and hours in very active glands. Autoradiographs showed that the 45Ca concentration was higher in association with the follicular epithelium than with the follicular lumen at 1 h or less after injection. However, at 24 h, the concentration of 45Ca was higher in the lumen than elsewhere in the gland. To determine rate constants describing the equilibration, the data in the normal gland were fitted with a model that took account of the differing rates of equilibration in cell and lumen. The presence of calcium in the lumen indicates that each time the follicular epithelial cell endocytosed stored luminal colloid before secretion, it also took in an appreciable amount of calcium.


Increase in pituitary levels of luteinizing hormone and follicle-stimulating hormone after pinealectomy in both intact and castrated male rats. Twenty-eight-day-old male Wistar rats were pinealectomized and/or castrated. Animals were killed 26 days later and anterior pituitaries were dissected out, weighed, and their content of LH and FSH measured by RIA. Pinealectomized rats showed higher body weights than sham-operated animals, which suggests an inhibitory influence of the pineal on growth. The weights of pituitaries were increased by both pinealectomy and castration. However, in pinealectomized rats, but not in castrated, the elevated pituitary weights seemed to be due to the high body weights of those animals. The pituitary content and concentration of LH and the content of FSH were increased by pinealectomy in both intact and castrated rats. This finding may suggest that the pineal gland exerts an inhibitory action on the hypothalamic-pituitary axis that seems to be independent of the testicular function.


Glucocorticoid binding by isolated lung cells. Synthesis of surfactant in the lung of fetal, and perhaps adult, animals responds to glucorticoids, and glucocorticoid receptor activity has been identified in this tissue of several sepecies. To determine whether receptor is present in the alveolar type II cell, which is the site of surfactant production, we studied glucocorticoid binding by various populations of lung cells. Specific binding was demonstrated in freshly isolated populations of rat lung cells containing primarily alveolar type II cells, in organotypic cultures derived from fetal rat lung containing 90% type II cells, in cultured A549, L-2, and F-42 cell lines which apparently originated from type II cells, and in human lung fibroblastic cells. The equilibrium dissociation constants for nuclear binding of dexamethasone by intact cells at 37 C ranged from 5.0--10.8 nM, and the number of binding sites per cell ranged from 5,700--57,000. In cytosol preparations from L-2 and A549 cells, there was equivalent specific binding of both natural and synthetic corticosteroids, and binding activity had the expected specificity for steroids with glucocorticoid activity. These findings indicate that glucocorticoid receptor is present in both fetal and adult pulmonary type II cells and in cell lines which apparently originated from these cells. The presence of receptor in type II cells in consistent with direct action of glucocorticoids on these cells in vivo.


Transformation of normal follicles into thyrotropin-refractory "cold" follicles in the aging mouse thyroid gland. Autoradiographs of thyroid glands of aging mice demonstrate the gradual appearance of "cold" follicles which fail to iodinate the intraluminar iodoproteins even after intense exogeneous or endogeneous TSH stimulation. "Cold" follicles first appear at the age of 5 months. They may account for 80% of all thyroid follicles in 13-month-old mice. Morphologically, the "cold" follicles are characterized by a larger than normal colloid volume and a comparatively flat epithelium for any given follicle size. Old thyroids are twice as large as young ones. They contain twice as much normally iodinated thyroglobulin/mg wet weight. The total number of cells per gland remains constant throughout the life time. This is probably also true for the total number of follicles. The iodide pump, as judged by the tissue to serum ratio, remains normal and TSH-responsive. Yet, it is not established whether this is due to a higher activity of the pump in normal follicles or to preservation of inorganic iodide transport in "cold" follicles. Pinocytosis is defective in "cold" follicles and is poorly responsive to TSH stimulation. Furthermore, diffusion of iodocompounds is severely impaired in the colloid of "cold" follicles. It is suggested that the primary defect in the pathogenesis of "cold" follicles in old mice is the gradual failure of endocytosis to respond to normal TSH stimulation. Because exocytosis and iodination would first proceed normally, the follicular lumina would become overdistended up to a point where the apical membrane is functionally impaired. With this sequence of events, "cold" follicles would have impaired iodination while being metabolically as active as hot ones. Thus, several puzzling reports on high metabolic activity of "cold" tissue could possibly be interpreted.


Effect of pregnant mare's serum gonadotrophin on the activities of delta 4-5 alpha-reductase, aromatase, and other enzymes in the ovaries of immature rats. After incubation of progesterone, 17 alpha-hydroxyprogesterone, androstenedione, and testostrone with an ovarian preparation (supernatant fluid at 10,000 x g) of immature rats (21-23 days of age) in the presence of NADPH, 3 alpha- and 3 beta-hydroxy-5 alpha-reduced steroids were obtained as the major metabolites. Among the enzyme activities relevant to the metabolism, delta 4-5 alpha-reductase and 3 beta-hydroxysteroid dehydrogenase were intracellularly localized to the microsomal fraction (10,000--105,000 x g), and 3 alpha-hydroxysteroid dehydrogenase was detected exclusively in the cytosol fraction (supernatant fluid at 105,000 x g). Within 2 days after a single injection of pregnant mare's serum gonadotrophin (10 IU/rat) to 21-day-old female rats, the following occurred: 1) an enhancement of 17 alpha-hydroxylase and C-17-C-20 lyase activities; 2) a suppression of delta 4-5 alpha-reductase activity; and 3) an increase in aromatizing activity. From the above-mentioned results, it was concluded that the increased secretion of estrogen from ovaries of immature rats stimulated by pregnant mare's serum gonadotrophin administration was caused by a modification of the ovarian enzyme activities relevant to estrogen production.


Relaxin bioactivity and immunoactivity in human corpora lutea. Relaxin-like activity in extracts of corpora lutea (CL) from pregnant and non-pregnant women was determined by radioimmunoassay and by guinea pig pubic symphysis palpation assay. The biologically determined activity paralled the immunoactivity of extracts of CL of pregnancy. The relaxin content of CL of non-pregnant women was too low for detection by the bioassay.


Thyroid hormone analogs and fetal goiter. Transfer of iodothyronine across the placenta in most species occurs only with difficulty. Recently, biologically active, nonhalogenated thyroid hormone analogs have been synthesized with properties which might favor placental transfer. To test this possibility, we compared the doses of T4, T3, and thyroid hormone analogs necessary to prevent propylthiouracil-induced goiter formation in rat fetuses. T4 and T3 prevented fetal goiter, but in doses that caused maternal hyperthyroidism; in contrast, the thyroid hormone analogs prevented fetal goiter in doses that were not thyrotoxic to the mother.


Effects of parathyroid hormone on the distribution and transport of calcium in cultured kidney cells. The effects of parathyroid hormone (PTH) on the transport and distribution of calcium in isolated kidney cells were studied by kinetic analysis of 45Ca desaturation curves. The results show that PTH (15 ng/ml) increases the total cell calcium content, every exchangeable pool, and all exchange rates. The greatest effect is observed in the slowest kinetic phase which reflects a mitochondrial pool. The effects of PTH occur even in the absence of magnesium and of phosphate and the larger effect is again obtained in the mitochondrial pool. These results suggest that PTH stimulates calcium transport in kidney cells by affecting the intracellular distribution of calcium or by stimulating calcium influx.


Studies on the mechanism of 3 alpha-androstanediol-induced growth of the dog prostate. The conditions under which dihydrotestosterone and 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-androstanediol) induce prostatic growth in the castrate dog have been investigated in animals in which the initial size of the prostate was either normal or large. When androgen replacement was commenced immediately after castration of dogs with preexisting prostatic hypertrophy, dihydrotestosterone was as effective as 3 alpha-androstanediol in maintaining prostate weight. Under all other conditions examined (replacement commenced immediately after castration in dogs with small prostates or 2 weeks after castration in dogs with large prostates), 3 alpha-androstanediol but not dihydrotestosterone induced significant prostatic growth. The maximal effect of 3 alpha-androstanediol in inducing prostatic growth was observed in 12 weeks. The administration of 17 beta-estradiol enhanced the growth-promoting action of 3 alpha-androstanediol, but did not affect the action of dihydrotestosterone or influence 3 alpha-hydroxysteroid dehydrogenase activity in prostate. Thus, the synergistic effect of 17 beta-estradiol and 3 alpha-androstanediol seems to be independent of any effect on the interconversion of the two androgens. The activity of the 3 alpha-hydroxysteroid dehydrogenase in dog prostate was high under all conditions of accelerated prostatic growth and appeared to be under androgenic control.


A direct effect of medroxyprogesterone acetate on 17 beta-hydroxysteroid dehydrogenase in adult rat testis. Sixty-day-old rats were divided into four groups and treated for 30 days with either medroxyprogesterone acetate (Provera), gonadotropins (bovine LH and ovine FSH), Provera plus gonadotropins, or saline. The progestin treatment resulted in a lowering of plasma levels of testosterone, androstenedione, and LH, as well as in a reduction of epididymal sperm counts and accessory sex organ weights. The progestin-treated groups showed markedly lower levels of testicular 17 beta-hydroxysteroid dehydrogenase activity (35% of controls) and delta 5,3 beta-hydroxysteroid dehydrogenase activity (70% of controls). Rats treated with only gonadotropins exhibited reduced 17 beta-hydroxysteroid dehydrogenase but increased delta 5,3 beta-hydroxysteroid dehydrogenase activities. It was concluded from these results that progestins may affect testicular steroidogenesis and spermatogenesis not only by reducing LH secretion but also by a direct effect on the testis, as LH suppression could not account for the inhibition of 17 beta-hydroxysteroid dehydrogenase activity. Long term progestin treatment did not alter the steroidogenic response of the testis to acute administration of LH, although the testosterone to androstenedione ratio in plasma was decreased.


Comparison of the effects of calcium and magnesium on parathyroid hormone secretion rate in calves. Parathyroid hormone (PTH) secretion rate was measured in seven calves by using a technique which involved RIA of parathyroid venous blood collected during timed intervals and measured volumetrically. Infusion of solutions of NA2EDTA and MgCl2 into the jugular vein was used to alter plasma calcium and magnesium concentrations. In four calves, elevation of plasma magnesium concentration rapidly decreased the PTH secretion rate that had been stimulated by induced hypocalcemia. In three calves, equimolar and opposite changes in plasma calcium and magnesium concentrations were induced by simultaneous infusions of Na2EDTA and MgCl2. Despite the equimolar increase in plasma magnesium concentration, PTH secretion was increased in response to the decline in plasma calcium concentration. In three experiments, the concentration of each cation was kept constant during periods in which the concentration of the other cation was varied. The effect of variations in plasma magnesium concentration on PTH secretion rate was approximately 1/3-1/2 as great as that observed with changes in plasma calcium concentration. These observations indicate that the effect of magnesium on PTH secretion rate is similar to that of calcium, but not equipotent.


Lack of effect of hypomagnesemia on elevated plasma 1,25-dihydroxycholecalciferol in acutely phosphate-deprived rats. The effect of hypomagnesemia occurring in phosphate deprivation on elevated levels of plasma 1,25-dihydroxycholecalciferol (1,25(OH)2D3) was evaluated in rats. Phosphate deprivation was associated with an elevated plasma 1,25(OH)2D3, hypophosphatemia, and hypomagnesemia. With oral magnesium supplement during phosphate deprivation, plasma magnesium did not fall, but plasma inorganic phosphate fell and 1,25(OH)2D3 rose to comparable levels seen in phosphate-deprived rats lacking magnesium supplement. These results indicate that a rise in plasma 1,25(OH)2D3 during phosphate deprivation is not due to concomitant hypomagnesemia and support the postulate that phosphate deprivation per se is responsible for the elevated plasma 1,25(OH)2D3.


Etiology of anovulation in the immature alloxan-diabetic rat treated with pregnant mare's serum gonadotropin: absence of the preovulatory luteinizing hormone surge. The effects of alloxan-induced diabetes on ovulation and other ovarian responses were investigated in immature rats injected with PMS gonadotropin (PMSG, 15 IU/100 g) on day 30 of age. Rats were killed on day 32 (presumed proestrus) or on day 33, at which time the oviducts were examined for ova. Ovarian weight gain was similar in control and diabetic rats and Graafian follicles were present in both groups on day 32. None of the diabetic rats ovulated while 96% of the control rats ovulated. Anovulation in diabetic rats could not be attributed to a drug side-effect of alloxan or to a lack of ovarian responsiveness, as 90% of the animals ovulated after treatment with insulin or with hCG (5 IU). Measurements of serum estradiol and LH on the morning of presumed proestrus revealed that concentrations of these hormones were not different in control and diabetic rats. However, measurements of LH in blood samples taken in the afternoon from control rats showed an LH surge, whereas no LH surge was found in diabetic rats. Thus, anovulation in immature diabetic rats treated with PMSG is not caused by an attenuation of ovarian responsiveness or by decreased secretion of estradiol, but rather is due to the loss of the LH surge.


Studies on the isolation and chemical-physical properties of porcine follicle-stimulating hormone. Methods are described for isolating highly purified FSH from porcine pituitary glands. The biological activity of pure material was 22 NIH-FSH-P2 U/mg, as judged by the ovarian weight augmentation bioassay. Virtually no immunoreactive LH was indicated by RIA, and only one component was detected by both gel electrophoresis and immunoprecipitation. Amino acid and carbohydrate analyses of the highly purified FSH indicated the presence of 1-tryptophan, a high content of aspartic and glutamic acids, and 6% sialic acid. Molecular weights of untreated and guanidine-treated porcine FSH were estimated from hydrodynamic measurements of Stokes radii and sedimentation coefficients. A highly specific and sensitive RIA was also developed for this hormone.


Antiserum to somatostatin reverses starvation-induced inhibition of growth hormone but not insulin secretion. The role of SRIF in starvation-induced inhibition of GH and insulin secretion was assessed by passive immunization with anti-SRIF serum. Six-hour secretory profiles obtained from chronically cannulated male rats deprived of food for 72 h showed marked suppression of GH secretory bursts and significant depression of plasma insulin levels. Administration of 1 ml SRIF antiserum (SRIF AS) iv to starved rats resulted in rapid (within 15 min) restoration of high amplitude GH pulses (600-800 ng/ml) and sighificant elevation of GH trough values. The mean 6-h GH level of starved SRIF, AS-treated rats (189.2 +/- 23.9 ng/ml) was significantly higher than that of starved, normal sheep serum-treated control animals (62.8 +/- 5.8 ng/ml) (P less than 0.005). In contrast to the effects on GH, plasma insulin levels in starved rats administered SRIF AS remained low. No significant difference was observed in the mean 6-h plasma insulin level of starved-SRIF, AS-treated rats when compared to starved, normal sheep serum-treated controls. These findings suggest that circulating SRIF is a physiological regulator of starvation-induced GH suppression but is not involved in mediating the inhibition of insulin.


Formation of DES-ASP1-angiotensin II is not an obligatory step in the steroidogenic action of angiotensin II in the canine adrenal. A comparison of the receptor binding properties and in vitro metabolism of angiotensin II and [des-Asp1]-angiotensin II was performed in collagenase-dispersed glomerulosa cells from dog adrenal glands. Each peptide was bound rapidly and reversibly to isolated glomerulosa cells, with similar equilibrium and kinetic constants of association and dissociation. The labeled peptide that was bound to glomerulosa cells after incubation with 125I- and 3H-angiotensin II at 37C for 30-60 minutes was predominantly angiotensin II, with about 15% [des-Asp1]-angiotensin II. These findings demonstrate that local formation of the heptapeptide is not an obligatory step in the action of angiotensin II on aldosterone production in the canine adrenals.


Serum triiodothyronine in undernourished rats: dependence on dietary composition rather than total calorie or protein intake. Serum triiodothyronine (T3) in rats did not change if food intake was restricted without altering the composition of the diet. Feeding a hypocaloric low-protein high-carbohydrate diet caused increases in serum T3, while feeding a hypocaloric high-protein low-carbohydrate diet caused decreases in serum T3. These changes were not related to reductions in intake of either calories or protein, and concomitant changes in serum thyroxine (T4) were not observed. Reduction in the dietary content of valine, without changes in the percentage of carbohydrate in the diet, caused increases in serum T3 when compared to growth-matched controls on a diet with normal valine concentration. Serum T3 in undernourished rats depends on the composition of the diet and not on total intake of calories or protein.


Adrenal steroidogenesis in methylandrostenediol-induced hypertension. Adrenal vein catheterizations were done in rats made hypertensive by administration of methylandrostenediol (MAD; 17alpha-methyl-5-androstene-3beta,-17beta-diol), and in control rats at intervals during treatment. All MAD-treated rats were hypertensive by 7 weeks. Secretion of corticosterone was consistently decreased at all times in MAD-treated rats. 18-Hydroxy-11-deoxycorticosterone secretion and 11-deoxycorticosterone (DOC) secretion decreased and increased, respectively, compared to controls at 2, 4, and 6 weeks. Aldosterone secretion was decreased at 2 and 4 weeks. This study shows an in vivo block of adrenal 11- and 18-hydroxylation. Transient DOC accumulation by treatment with MAD produced hypertension, though DOC oversecretion and other changes in steroidogenesis were waning by the time hypertension developed.


The distribution of biologically active and inactive radioiodinated human calcitonin in the rat. The anatomical distribution of a biologically active and biologically inactive preparation of radioiodinated human calcitonin (hCT) was studied in rats. The former was prepared by electrolysis and the latter by chloramine-T oxidation. When administered iv, the distribution of the two species of [125I]hCT was nearly identical. The kidney was the principle organ of distribution for the hormone while less was present in liver, intestine, or bone. In vitro, kidney homogenates rapidly degraded [125I]hCT. This degradation was partially blocked by trypsin inhibitor and fully blocked by ACTH at both 4 and 37 C. These studies demonstrate the importance of kidney in the metabolism of calcitonin and suggest that the in vivo distribution of biologically active and inert species of the hormone may be indistinguishable.


Experimental hypertension and other responses to 18-hydroxy-deoxycorticosterone treatment in the rat. Young female unilaterally nephrectomized, salt-loaded, Sprague-Dawley rats were treated with 200 microgram or 1 mg 18-hydroxy-deoxycorticosterone-21-acetate (18-OH-DOCA) in oil daily, and a group of kidney-intact animals on a normal salt intake was given 2 mg/day. The hormone was not found to increase saline consumption, increase urinary potassium or kallikrein excretion, or depress serum renin activity or potassium concentration. Slight hypertension did develop at 3 weeks in salt-loaded rats on the lowest dose, but this was neither increased by higher dosage or longer treatment, nor reflected by increased heart or kidney weight. The effect of 40-mg pellet implantation of DOCA and 18-OH-DOCA was then compared in unilaterally nephrectomized, salt-loaded, female Fischer 344 rats. The former caused increased saline consumption, hypertension, hypokalemia, and heart and kidney enlargement, whereas 18-OH-DOCA did not. Thus, the hypertensogenic potency of 18-OH-DOCA is, at best, a reflection of its known, very weak, mineralocorticoid activity.


Central control of hepatic steroid metabolism: effect of discrete hypothalamic lesions. The effects of various hypothalamic lesions on hepatic steroid metabolism in adult rats were investigated. It was found that frontal deafferentation at the retrochiasmatic and suprachiasmatic level resulted in a complete "feminization" of hepatic steroid metabolism in male rats. Such an effect was also seen when lesions involving mainly the anterior periventricular hypothalamic area and the suprachiasmatic nucleus were performed in male rats. Midline lesions, anterior to the suprachiasmatic nucleus, on the other hand, did not result in any significant effects. A moderate degree of "feminization" was obtained after bilateral lesions involving mainly the nucleus interstitialis striae terminalis but including also parts of the anterior commissure. Small lesions in the lateral preoptic area were, however, without effect. No effects were seen of analogous lesions in female rats in any of the cases studied. The present findings suggest that a region including the anterior hypothalamic periventricular area, the suprachiasmatic nucleus and adjacent areas is involved in the control of hepatic steroid metabolism. It is postulated that the neuronal cell bodies that produce a factor with an inhibitory effect on the secretion of "feminizing factor" have their origins in this area of the hypothalamus, or, alternatively, may send axons through this area to the basal hypothalamus and thus directly or indirectly influence the anterior pituitary gland.


Stimulation of 18-hydroxy-11-deoxycorticosterone secretion by angiotensin II and potassium. The control of 18-hydroxy-11-deoxycorticosterone (18-OH-DOC) secretion is incompletely understood: ACTH seems to be the dominant regulator, the importance of angiotensin II (A-II) is uncertain, and the effect of potassium has not been investigated. The purpose of this study was to evaluate the 18-OH-DOC response to these three stimuli in vitro. Suspensions of isolated rat adrenal glomerulosa or fasciculata cells were stimulated with either alpha-1-24 ACTH (0.04 mU/ml), A-II (25 ng/ml), or potassium (5.9 mEq/liter), and 18-OH-DOC production was measured. In glomerulosa cells, ACTH produced the greatest 18-OH-DOC response but A-II and potassium also produced significant (P less than 0.001) 18-OH-DOC increases (control, 162 +/- 14 (SE) ng/10(6) cells incubated; A-II, 368 +/- 39; potassium, 380 +/- 37; ACTH, 1544 +/- 165). In fasciculata cells, 18-OH-DOC production increased with ACTH but not with A-II or potassium. These results document that A-II and potassium, as well as ACTH, can stimulate 18-OH-DOC production by glomerulosa but not by fascicuata cells. The response to A-II may provide an explanation for the reported increase in 18-OH-DOC production after sodium restriction.


Changes in the fetal thyroid axis after induction of premature parturition by low dose continuous intravascular cortisol infusion to the fetal sheep at 130 days of gestation. The effect of cortisol infusion on plasma T4 and T3 concentrations has been investigated in the fetal lamb at 130-134 days of gestation. Infusion of cortisol at rates equivalent to one-third the quantity produced by the fetus at the time of parturition results in a significant fall in fetal plasma T4 concentration and a significant rise in fetal plasma T3 concentration. Delay in the initiation of labor as a result of progesterone administration to the ewe did not prevent the changes in the fetal thyroid axis. These modifications of thyroid function therefore appear to be caused by the infusion of cortisol rather than to be secondary to the induction of labor.


Direct evidence that the arcuate nucleus-median eminence tuberoinfundibular system is not of primary importance in the feedback regulation of luteinizing hormone and follicle-stimulating hormone secretion in the castrated rat. Adult rats which have received monosodium-L-glutamate (MSG, 4 mg/g BW) on alternate days for the first 10 days of life manifest central nervous system lesions largely restricted to the retina and the arcuate nucleus of the hypothalamus in which nearly 90% of the perikarya are destroyed, leaving axons in passage intact. In animals so treated, concentrations of dopamine within the arcuate nucleus and median eminence of the hypothalamus are reduced 52% and 57%, respectively, in males and 45% and 61% in females, whereas concentrations of norepinephrine in these same two areas are normal. Concentrations of both norepinephrine and dopamine in five other hypothalamic nuclei (dorsal septal, medial preoptic, suprachiasmatic, periventricular, and dorsomedial nuclei) are unchanged. Nevertheless, despite the destruction of the arcuate nucleus cell bodies of MSG-treated rats, postcastration levels of serum FSH and LH in males, and FSH in females were not significantly different from FSH and LH values in castrated controls. Serum LH in castrated, MSG-treated females was slightly but significantly lower than in castrated controls. It is concluded that the arcuate nucleus-median eminence tuberoinfundibular neurons are not of primary importance in the tonic, negative feedback regulation of gonadotropin secretion.


The biphasic regulation of prolactin secretion by dopamine agonist-antagonists. The in vitro secretion of PRL by the pituitary gland is inhibited by low concentrations of dopamine. Dopamine receptor-blocking agents, haloperidol and pimozide, in concentrations between 10-100 nM, neutralize virtually completely the dopamine-mediated inhibition of PRL secretion. At these concentrations, the dopamine antagonists alone are without effect on PRL secretion. Higher concentrations of these agents, however, have a powerful action to inhibit PRL secretion. Perphenazine was without direct effect on the in vitro secretion of PRL. These observations may be of general importance to the study of dopamine action in other organ systems.


Evidence for a discontinuous requirement for estrogen in stimulation of deoxyribonucleic acid synthesis in the immature rat uterus. Estriol is rapidly lost from uterine target cell nuclei. Therefore, it has been used to study the relative time of exposure to estrogen that is necessary to elicit the late growth responses of increased DNA polymerase activity or increased rate of DNA synthesis observed after 17beta-estradiol treatment. Estriol administered during a second critical phase from 9-15 h after the initial injection elicits maximal DNA synthesis. Our results suggest that there is a discontinuous requirement for estrogen in the sequence of events which result in the uterine growth response to estrogenic hormones. If estriol is present during critical phases of this sequence of events, it is equipotent to estradiol in eliciting the full uterotropic response.


Subcellular distribution of thyroxine 5'-deiodinase in the rat kidney: a plasma membrane location. The enzymatic activity in rat kidney homogenates mediating the conversion of T4 to T2 has been previously shown to be particulate. The subcellular organelle responsible for T4 5'-deiodination, however, has not been identified. Enzymatic activity was assessed with outer ring 125I-labeled L-T4 as the substrate and the iodothyronines were separated by descending paper chromatography. Comparison of the distribution of T4 5'-deiodinase activity with that of established enzyme markers of subcellular organelles demonstrated: 1) T4 5'-deiodinase activity paralleled that of plasma membrane enzyme markers in subcellular fractions prepared by differential centrifugation, with 80% of the T4 5'-deiodinase and (Na+/K+)-ATPase equally distributed in the crude nuclear and microsomal fractions; 2) the smooth microsomal fraction, known to be contaminated with plasma membrane in kidney tissue preparations, contained the bulk of the T4 5'-deiodinase activity and plasma membrane marker enzymes found in the microsomal fraction; and 3) purified plasma membrane showed enrichment of T4 5'-deiodinase comparable to that of the known plasma membrane enzyme (Na+/K+)-ATPase. These data suggest that T4 5'-deiodinase in kidney tissue is an enzyme of the plasmalemma.


The subcellular localization of the long-acting thyroid stimulator inhibitor in bovine thyroid gland. To characterize further the nature and site of the receptor for the IgG stimulator in Graves' disease, we have developed a preparative scheme to provide a bovine thyroid subfraction rich in plasma membranes. Pellets (800 X g) obtained from bovine thyroid homogenates were layered on a 30-64% continuous sucrose gradient (SG). The centrifuged gradient was divided into four portions (SG1, 2, 3, and 4); and these along with the 800 X g pellet were characterized in terms of their capacity to neutralize (bind) the biological activity of LATS, their specific binding of [125I]TSH, and their subcellular marker content. Subfraction SG1 contained the highest adenyl cyclase specific activity, the highest [125I]TSH binding specific activity, and vied with the 800 X g pellet and SG3 for the highest specific activity of LATS neutralization. Electron microscopy of SG1 showed a predominance of plasma membrane structures contaminated with a modest amount of cellular debris. Adenyl cyclase activity in SG1 was enhanced by TSH, LATS, and sodium fluoride. Although a dose-response curve could be established for TSH, a similar relationship could not be established for LATS. We conclude that activation of plasma membrane-bound adenyl cyclase is associated with neutralization of the biological activity of LATS. Further, the difference between [125I]TSH binding and LATS neutralization activity observed in the various thyroid membrane subfractions suggests that either LATS and TSH interact at different sites or have different mechanisms of binding at a common site.


The maturation of rabbit epididymal spermatozoa in organ culture: inhibition by antiandrogens and inhibitors of ribonucleic acid and protein synthesis. Infertile spermatozoa from the proximal corpus epididymidis will become fertile when this epididymal segment is cultured 24 h in vitro with 5alpha-dihydrotestosterone (5alpha-DHT). The effect of antiandrogens and inhibitors of RNA and protein synthesis upon this 5alpha-DHT-induced sperm maturation was investigated. The response to 5alpha-DHT was abolished by cyproterone acetate, SKF 7690, actinomycin D, cycloheximide, puromycin, and the aminonucleoside analog of puromycin. Addition of cyproterone acetate or actinomycin D to a suspension of distal corpus spermatozoa did not change their fertilizing ability. Culture of segments of the distal corpus for 24 h with the same antiandrogens and RNA and protein synthesis inhibitors did not change the fertilizing ability of spermatozoa. These results indicate that the development of the sperm-fertilizing ability observed in the proximal corpus epididymidis in vitro in response to 5alpha-DHT is dependent upon binding of 5alpha-DHT and synthesis of new RNA and protein molecules by the target cell. They also indicate that antiandrogen and inhibitors of RNA and protein synthesis do not have a nonspecific toxic effect on spermatozoa during the time period studied. It is likely that the target cells are the epididymal epithelial cells rather than the spermatozoa themselves.


Evidence that the effects of isoproterenol on water intake and vasopressin secretion are mediated by angiotensin. The effect of isoproterenol (6 microgram/kg sc) on drinking, urine flow, and vasopressin secretion was examined in a group of trained dogs with chronically implanted third ventricular cannulae. Isoproterenol stimulated drinking in association with a reduction in urine flow and an increase in urine to plasma osmolality ratio. Plasma renin activity increased from 3.1 +/- 0.8 to 13.0 +/- 2.7 ng/ml/3 h and plasma vasopressin concentration increased from 11.3 +/- 1.3 to 40.3 +/- 12.5 pg/ml. The effect of isoproterenol was reexamined during an intracerebroventricular infusion of the angiotensin II antagonist, saralasin (0.02 microgram/kg/min). This treatment did not affect the isoproterenol-induced increase in plasma renin activity, but inhibited the drinking, antidiuresis, and increase in plasma vasopressin concentration. These data indicate that the effects of isoproterenol on drinking, urine flow, and vasopressin secretion are mediated via the renin-angiotensin system.


Prolonged suppression of insulin release by a somatostatin analog. A somatostatin analog, [D-Ala5, D-Trp8]-somatostatin, has been found to selectively inhibit insulin and GH release in rats. The release of these hormones is inhibited by an analog dose of 5 microgram/kg in short term experiments (15 min from analog administration to blood sampling), while glucagon levels are not lowered by analog doses as high as 500 microgram/kg. The lowered insulin to glucagon ratio results in hyperglycemia. [D-Ala5, D-Trp8]Somatostatin is also long acting; a 1 mg/kg dose results in hypoinsulinemia for 2 h and hyperglycemia for 3 h.


In vitro decidualization of rat endometrial cells. Rat endometrial cells were isolated from ovariectomized adults submitted to a progesterone-estradiol sequence proper to sensitize the uterus to respond to a deciduogenic stimulus, and then cultured in vitro in the presence of progesterone. They grew rapidly and became morphologically similar to the decidual cells occurring in vivo with regard to the binucleation and the accumulation of characteristic filamentous material in the cytoplasm. The life-span of the decidualized cells in vitro was comparable to the duration of the deciduoma in vivo. The progesterone treatment of the rat was found to be a prerequisite for such development and transformation of endometrial cells in vitro, since control experiments performed with nonhormonally injected castrated animals gave only isolated islets of epithelial cells and scattered fibroblast-like cells in culture, whereas the decidual transformation was still observed, although with some delay, with cells from progesterone (but not estradiol)-treated spayed rats. Such an in vitro system may provide a new opportunity to study the mechanism of decidualization and of its hormonal control.


Metabolic clearance of endogenous and radioiodinated thyroglobulin in rats. The basal concentration and metabolic clearance of radiolabeled and endogenous thyroglobulin (Tg) were measured in normal male rats. Serum Tg concentrations (by RIA) in Sprague-Dawley adult males ranged from 70-680 ng/ml (mean +/- SD, 218 +/- 177). In male Fisher rats, basal serum Tg concentrations were 32-263 ng/ml. The fractional disappearance rate of endogenously radioiodinated rat Tg and of endogenous rat Tg after thyroidectomy were both about 8%/h. The MCR of endogenously labeled Tg was 2.4 +/- 1.1 ml/100 g/h. Using this clearance and the mean serum Tg concentration in these rats, a daily thyroidal release rate of about 201 microgram Tg/100 g BW would be required to provide the T4 metabolized daily by the rat, the Tg released into the circulation comprises about 7% of the total rat Tg produced in a 24-h period.


The effect of epinephrine on phenylethanolamine N-methyltransferase in cultured explants of adrenal medulla. The investigation reported here was designed to gain further insight into the mechanisms by which phenylethanolamine N-methyltransferase (PNMT) is regulated. Explants of adrenal medullae were cultured in defined media for up to 48 h, during which time the tissue remained histologically intact. Addition of epinephrine to the medium led to a diminution in the activity of PNMT, measurable in the dialyzed homogenates of the cultured tissue. The enzyme activity was inversely proportional to the concentration of epinephrine present in the culture medium. A diminution in the amount of PNMT protein also resulted from incubation of the explants in the presence of epinephrine. The extent of loss of PNMT, measured by immunochemical titration, corresponded to the degree of loss in PNMT activity under the same conditions. None of the metabolites of epinephrine, including 3,4-dihydroxy- or 3-methoxy-4-hydroxymandelate, or metanephrine, had an effect on PNMT. Tyrosine hydroxylase and catechol O-methyltransferase activities were also diminished, whereas tyrosine transaminase, acid phosphatase, and monoamine oxidase activities were not affected by addition of epinephrine to the medium. Ascorbic acid added to the medium prolonged the lifetime of epinephrine but did not alter the degree of diminution of PNMT. The results obtained are consistent with the view that epinephrine is rapidly assimilated into the cytoplasm of medullary cells and plays an important role in regulating the concentration of PNMT in the adrenal gland.


Prolactin suppression by (-) delta-9-tetrahydrocannabinol (THC): involvement of serotonergic and dopaminergic pathways. (-) delta-9-Tetrahydrocannabinol (THC) was previously shown to suppress serum PRL levels (SPL) in rats. In the present study, various pathways by which THC suppresses PRL secretion were investigated. THC abolished the elevated SPL induced by either 5-hydroxytryptophan or melatonin. The SPL suppression after THC treatment was abolished upon treatment with cyproheptadine. These results suggest on involvement of a serotonergic pathway in the suppressive effect of THC on PRL secretion. Elevated SPL induced by a low dose of the dopaminergic blocker pimozide was suppressed after treatment with THC. However, elevated SPL, induced by a high dose of pimozide or by any dose of perphenazine (a less specific dopaminergic blocker), were not reduced by combined treatment of either of these drugs with THC. It seems that THC also enhances the dopaminergic activity in the pathway that controls PRL secretion. In addition, the pimozide-induced SPL elevation dropped upon combined treatment of cyproheptadine and pimozide. As cyproheptadine alone, and in the dose used, did not affect SPL, it is proposed that the serotonergic and dopaminergic pathways that control PRL secretion do not necessarily act independently of one another.


Effects of a single injection of estradiol valerate on the hypothalamic arcuate nucleus and on reproductive function in the female rat. Young adult cyclic female rats were each injected with 2 mg estradiol valerate (EV) in sesame oil. Controls received an equivalent volume of sesame oil. Within 2 months after injection, most of the EV-treated animals showed persistent vaginal estrus and small polyfollicular ovaries as well as pathological changes in the hypothalamic arcuate nucleus. This pathological process was gradually progressive such that by 6 months after EV injection, the basal lateral region of the nucleus contained numerous reactive microglia, astrocytes, and degenerating elements of the neuropil. The experimental rats had elevated plasma PRL and GH concentrations which gradually diminished. Plasma estradiol concentration remained elevated 2 months after injection, while plasma LH and FSH concentrations stayed within the high and low normal range, respectively. The pituitary glands of injected animals weighed significantly more than those of controls 5.5 months after injection, but the enlarged glands did not cause hypothalamic compression. As mechanical anterior deafferentation of the medial basal hypothalamus has previously been shown to produce similar endocrine and reproductive alterations, it may be that estradiol treatment results in a functional-anatomical disconnection of the arcuate nucleus from the more anterior hypothalamic areas that regulate cyclicity. Whether this type of functional-anatomical phenomenon underlies other varieties of induced or secondary acyclicity in females remains to be determined.


Down regulation of insulin receptors in primary cultures of adult rat hepatocytes in monolayer. Down regulation of insulin receptor number by insulin at physiological concentrating (10(-9) M) has been demonstrated in primary cultures of adult rat liver parenchymal cells in monolayer. However, because insulin is rapidly degraded in this culture system, special efforts are necessary to maintain the concentration of insulin in the medium in order to evoke the down regulation phenomenon.


Prolactin release by vasoactive intestinal polypeptide in rats. Synthetic vasoactive intestinal polypeptide (VIP) administered either intraventricularly or iv caused a significant and dose-related increase in plasma PRL levels in urethane-anesthetized rats. The administration of naloxone, an opiate receptor antagonist, significantly blunted the plasma PRL response to VIP. Increases in plasma PRL induced by VIP were also significantly suppressed by L-dopa, a precursor of dopamine, whereas pilocarpine, a cholinergic agonist, diphenhydramine, a histamine antagonist, and cyproheptadine, an antiserotoninergic agent, did not affect the plasma PRL response to VIP. In in vitro experiments, VIP alone did not stimulate PRL release from cultured pituitary cells, but it significantly attenuated the inhibitory action of dopamine, which was not blocked by naloxone. These results suggest that VIP stimulates rat PRL secretion, at least in part, through activation of an opiate receptor in the central nervous system and by blocking the inhibitory action of a dopaminergic mechanism at the pituitary level.


Maternal, fetal, and amniotic fluid transport of thyroxine, triiodothyronine, and iodide in sheep: a kinetic model. A mathematical model of iodine kinetics in maternal and fetal sheep has been developed by combining separate iodide, T3, and T4 subsystems. The individual subsystem models were developed from literature studies of maternal-fetal exchange under thyroid-blocked and unblocked conditions. Rates of exchange, concentrations, and spaces of distribution were calculated by the SAAM computer program. The models for each of the subsystems required exchange compartments within the mother and fetus, exchanges between maternal and fetal circulations, and between the fetus and amniotic fluid. The fetal-amniotic fluid exchange was observed directly for iodide and indirectly for T3 and T4. No exchange between mother and amniotic fluid was required. It is possible that the amniotic fluid acts as a reservoir for these and other substances. Maternal-fetal kinetics suggest that low fetal T3 levels are maintained by an active transport of T3 from fetus to mother, a decreased transport from mother to fetus, and a low fetal T3 production. The model also requires that all fetal T3 loss occur via transport to the maternal system rather than via fetal utilization. In contrast, the fetal T4 system is largely autonomous, the small maternal exchange not significantly contributing to the fetal T4 economy. Fetal iodide seems to be supplied by a facilitated bidirectional exchange with the mother.


Effect of castration and steroid replacement on immunoreactive luteinizing hormone cells in the pars tuberalis of the rat. The response of LH cells in the pars tuberalis of the rat hypophysis to castration alone or castration and subsequent administration of testosterone propionate or estradiol benzoate was studied. To accomplish this, LH cells were labeled by the peroxidase-antiperoxidase immunocytochemical method, with use of an antiserum generated against hCG. LH cells were detected in the caudal region of the pars tuberalis of all intact control animals studied. After castration, LH cells in both the pars distalis and pars tuberalis showed similar degrees of hypertrophy and hyperplasia. This response was inhibited by either testosterone propionate or estradiol benzoate in the pars distalis as well as in the pars tuberalis. These results indicate that LH cells in the pars tuberalis, in addition to those in the pars distalis, are responsive to fluctuations in circulating steroid hormone levels and that the pars tuberalis may contribute to the overall secretion of gonadotropin by the adenohypophysis.


Interaction of gastric inhibitory polypeptide, glucose, and arginine on insulin and glucagon secretion from the perfused rat pancreas. Gastric inhibitory polypeptide (GIP) produced an increase in immunoreactive insulin (IRI) and immunoreactive glucagon (IRG) release from the isolated perfused rat pancreas. The magnitude of both effects was shown to be dependent on the prevailing glucose concentration. GIP stimulated IRG release at glucose concentrations less than 5.5 mM and IRI release at glucose levels greater than 5.5 mM. Arginine"stimulated IRG secretion in the presence of low glucose (2.7 mM) was potentiated by GIP. In contrast, GIP augmented arginine-stimulated insulin release only in the presence of arginine concentrations of (less than 20 mM, producing no further increase over a maximal arginine stimulus. The glucagonotropic effect of GIP in the presence of arginine was found to be suppressed by glucose, with the opposite effect observed with insulin release. It was concluded that the endocrine pancreatic action of GIP depends to a great degree on existing levels of modulating nutrients in the blood.


Comparison of pituitary-thyroid maturation in the fetuses of rats fed an iodine-deficient or normal diet. From the earliest detectable development of fetal pituitary-thyroid function (day 18-19 of gestation) through the first postnatal day, there was a higher degree of stimulation of the pituitary-thyroid axis in the fetuses of rats fed a low iodine diet (LID) than in those of rats fed a high iodine diet (HID). Significant differences between the two groups were consistently observed in relative thyroid size, plasma TSH, 4-h thyroid radioiodine uptake, and the labeled iodoamino acid composition of thyroid digests. Plasma T4 concentration was lower in both LID and HID fetuses and pups than in the HID mothers. Plasma T3 was not detectable (less than 20 ng/dl) in the fetuses of either group, nor was labeled T3 in the thyroid digests. Body weight, plasma T4, and pituitary TSH content were usually lower in the LID than the HID animals of comparable age; however, these differences were not consistently statistically significant (P less than 0.05). We conclude that iodine deficiency causes a marked stimulation of TSH secretion and, consequently, of thyroid growth and metabolism from the earliest development of fetal pituitary-thyroid function.


Inhibition by adenosine of thyroidal T4 release in vitro. Adenosine, like catecholamines, inhibits the thyroidal T4 release in vitro, when stimulated by TSH,N,O'-dibutyryl cyclic AMP [(Bu) 2cAMP], and phosphodiesterase inhibitors. Unlike catecholamines, the adenosine-induced inhibition is independent of adrenergic receptors. It is postulated that TSH stimulates thyroidal T4 release through a cAMP activated, adenosine-sensitive, protein kinase.


Analysis of luteinizing hormone and follicle-stimulating hormone release kinetics during a dynamic secretory event, the postpartum preovulatory surge in the rat, based on quantitative changes in stored and circulating luteinizing hormone and follicle-stimulating hormone and metabolic clearance data for these hormones. The original objective of this study was to use available distribution volume (V) and deca constant (k) estimates for rat LH and FSH to determine the amounts of these hormones synthesized and released during a dynamic secretory event, the postpartum preovulatory surge. The problem was approached by 1) determining the approximate rates and durations of pituitary LH and FSH depletion during the surge, and 2) comparing observed plasma LH and FSH patterns with patterns calculated on the assumptions a) that the rates and durations of LH and FSH release would have to equal or exceed the rates and durations of LH and FSH depletion, and b) that the V and k estimates reported by Bogdanove and Gay (Endocrinology 84: 1118, 1969) would be applicable in this situation. The observed surges proved to be 3- to 10-fold smaller than the calculated patterns, suggesting either that depletion exceded release or that inappropriate values of V and/or k had been used in the calculations. Subsequent reexaminations of LH clearance kinetics (Campbell et al., accompanying reports) provided larger estimates for both VLH and kLH. Recalculation of the data, using these new parameters, eliminated the apparent discrepancy between depletion and release. On the basis of these direct comparisons of stored and circulating LH, it seems that a "half-life" shorter than 30 min and a distribution volume greater than 3 ml/100 g must be used to model the effects of variations in LH release on circulating LH in the rat.


Prolonged infusion of rat luteinizing hormone alters its metabolic clearance pattern: indirect evidence for postsecretory mutation of luteinizing hormone. Constant rate infusion/stop-entry experiments of varied duration were done with rat pituitary LH to test the possibility that the relatively slow disappearance of endogenous circulating LH after hypophysectomy/stop-entry in orchidectomized rats might be due to redistributive distortion consequent to a prolonged "packing" of the extravascular LH spaces in these animals. The findings indicated that prolongation of LH infusion did result in progressive flattening of the decay curve after stop-entry. After 1 h of infusion, constant rate infusion/stop-entry decay of LH was nearly as rapid as decay after simple pulse injection of the same extract. After 24 h of infusion, it was nearly as slow as after hypophysectomy/stop-entry. However, this change could not be explained solely on the basis of redistribution, because a similar difference was seen when disappearance of "infused" and "injected" pituitary LH were examined by pulse injection of sera collected from rats which had been given this LH by injection or prolonged infusion. Although variations in extravascular LH "packing" might have influenced LH disappearance in the donor rats, there was no opportunity for such variations to develop in the recipients. Although the mechanism(s) involved in this progressive change in the metabolic clearance characteristics of exogenous (extracted) circulating LH might simply be selective accumulation of the molecules most capable for surviving in the blood, other possibilities exist (e.g. actual mutation of circulating LH molecules). Whether endogenous (secreted) LH undergone similar changes during its sojourn in the circulation is not yet known but these findings suggest that postsecretory mechanisms could contribute to the apparent qualitative difference(s) between stored and circulating rat LH. Several implications of this new information have been discussed.


Effect of hypophysectomy of cholesteryl ester synthesis and hydrolysis in testes and on serum lecithin-cholesterol acyltransferase activity in rats. Studies are reported of the effect of hypophysectomy on cholesterol esterase activity of testicular tissue and serum lecithin-cholesterol acyltransferase (LCAT) activity in rats. The testes of male Sprague-Dawley rats of 200-255 g were excised from animals sacrificed at 3, 7, and 15 days after hypophysectomy. Assays for cholesterol-esterifying and hydrolytic activities of the testicular tissues of these animals, compared to control animals, showed that hypophysectomy decreased both cholesteryl ester synthesis and hydrolysis. Hydrolytic activity was affected to a greater extent than esterifying activity. LCAT activity was significantly decreased by hypophysectomy compared to that of control animals. Although serum LCAT and testicular cholesterol esterase activities were decreased, the overall effect of hypophysectomy produced an increase in the level of serum cholesterol and cholesteryl esters. It is suggested that the role of essential fatty acids (EFA) in testicular function is related to the utilization of cholesteryl esters in androgen synthesis.


Data on the sites of the stimulatory feedback action of gonadal steroids indispensable for follicle-stimulating hormone release in the rat. The sites of the stimulatory feedback action of gonadal steroids on FSH release were examined in estrogen-primed ovariectomized rats. Estradiol benzoate (E2) injected 72 h after E2 priming induced a significant increase in serum FSH 6 and 30 h later. However, injections of estrone failed to induce a similar significant increase. Progesterone (P) injections also increased serum FSH 6 h later. Horizontal sections placed above the medial preoptic area prevented the increase in serum FSH after E2 injections but not after P injections. Furthermore, retrochiasmatic sections or bilateral lesions in the medial basal part of the suprachiasmatic area prevented the increase after E2 injections and P injections. E2, implanted into the bed nucleus of stria terminalis and the anterior hypothalamic area in E2-primed ovariectomized rats, induced a significant increase and decrease in serum FSH, respectively, whereas P implanted into the same areas failed to induce it. On the other hand, P implantations into the diagonal band of Broca and the lateral and the medial septum were effective in increasing and decreasing serum FSH, respectively, whereas E2 implantations into these areas were ineffective. Neither E2 nor P implanted into the medial amygdala, the medial preoptic area, or the hippocampus altered serum FSH levels. These results suggest that the stimulatory feedback actions of E2 and P on FSH release are exerted at different sites in the limbic structures.


Physical properties of androgen receptors in brain cytosol from normal and testicular feminized (Tfm/y hermaphrodite) mice. Our previous results had shown that brain cytosol from androgen-insensitive, testicular feminized (Tfm/y) mice contains a reduced amount (20-25%) of androgen receptors, compared to normal female or castrated male mice, with unchanged affinity for dihydrotestosterone (DHT). We have now used various physicochemical techniques to ask whether there is a qualitative difference between these residual receptors and those in normal brain. When androgen receptors labeled with [3H]DHT in concentrated crude brain cytosol were analyzed by density gradient centrifugation and Agarose gel filtration in buffers containing 0.4-0.5 M KCl, Tfm/y receptors appeared to be smaller and more symmetrical than those from their normal siblings; however, if wild-type receptors were partially purified or prepared from slightly more dilute homogenates, their properties approached those of Tfm/y receptors in crude cytosol. This suggested that receptor molecules in concentrated cytosol from normal mice were aggregated to a greater extent than were those from the mutant. The molecular weight (54,000) and axial ratio (3:1 for prolate or oblate ellipsoid) calculated for Tfm/y receptors, therefore, may provide estimates for normal receptors as well. By DNA-cellulose chromatography, Tfm/y and female cytosol receptors were both resolved into two components, eluting at about 0.13-0.15 and 0.22-0.24 M NaCl. On DEAE-cellulose columns, both were eluted as a single major species at approximately 0.08-0.10 M KCl. Thus, excluding their state of aggregation, presumably resulting from their different concentrations in crude cytosol, Tfm/y and normal receptors were substantially identical as concerns the physical parameters examined in this study.


Somatostatin content of the median eminence in female rats with lesion-induced disruption of the inhibitory control of growth hormone secretion. These experiments were designed to determine whether brain lesions which elevate nonstress plasma levels of GH and disrupt stress-induced suppression of GH secretion in female rats affect the median eminence content of somatostatin. Some rats received lesions in the medial or lateral preoptic-anterior hypothalamic area (PO-AHA), while others received anterior hypothalamic cuts. Sham-operated and intact rats served as controls. GH and somatostatin were measured by RIA. Medial but not lateral PO-AHA lesions caused elevated nonstress plasma GH levels at 2, 14, 17, and 23 weeks after surgery, but normal levels were obtained at autopsy at 27 weeks. These lesions compromised GH responses to stress at 14 and 23 weeks. Rats with anterior hypothalamic cuts showed elevated nonstress GH levels at 17, 23, and 27 weeks after surgery and loss of the GH response to stress at 14 and 23 weeks. Median eminence content of somatostatin was reduced approximately 80% in rats with medial PO-AHA lesions or anterior cuts. Whereas medial PO-AHA lesions were associated with normal body length and weight and evidence of estrogen secretion, anterior hypothalamic cuts produced increased linear growth and body weight and signs of functional castration. These results suggest that the effects of lesions which cause prolonged elevation of nonstress GH levels and disruption of the GH stress response are due to interference with somatostatin neurons located in the medial PO-AHA. Somatostatin content of the median eminence seems to depend largely on connections originating in or traversing the medial PO-AHA.


Metabolism of thyroid hormones by rat thyroid tissue in vitro. Rat thyroid lobes or hemilobes have been incubated in Krebs-Ringer phosphate buffer containing labeled T4 and/or T3, and the products were separated by paper chromatography. Labeled T4 was actively degraded; about half of the T4 metabolized was recovered as T3. Labeled T3 was also metabolized, but less rapidly than T4. Other than T3 produced from T4, the major products from both hormones were inorganic iodide and iodoprotein; the latter was presumably a secondary product of iodide organification because its formation was inhibited by hypoxia and methimazole. Feeding the animals a low iodine diet increased their hormone-metabolizing activity. Incubation under nitrogen did not affect the rate of T4 degradation, but partially inhibited T3 degradation. Degradation of both hormones was unchanged in the presence of methimazole and ascorbate, was markedly inhibited by 1 mM propylthiouracil (PTU), and was partially inhibited by azide and cyanide. Thyroid tissues concentrated both hormones, tissue to medium gradients averaging 5.4 for T4 and 20.7 for T3; none of the conditions affecting hormone degradation (incubation under nitrogen or with azide, cyanide, or PTU) significantly altered these gradients. It is concluded that the thyroid can metabolize both of its major hormones by a system distinct from thyroidal peroxidase. Hormone metabolism, therefore, is a potentially important factor in net hormone secretion. In its resistance to hypoxia, methimazole, and ascorbate and its sensitivity to PTU, the thyroid's system for generating T3 from T4 resembles T3-forming systems of liver and kidney. The thyroid, because T3 formation is its dominant pathway for T4 metabolism, may provide a useful model for study of this reaction.


Extraction of erythropoietin from normal kidneys. Significant amounts of active erythropoietin were extracted from the kidneys of normal rats, cattle, dogs, and rabbits by homogenization of the organs in 0.1 M phosphate buffer. The mean erythropoietin activities of the extracts, as determined by the starved-rat assay, were 0.26 U/g beef kidney, 0.41 U/g dog kidney, and 0.11 U/g rat kidney. The dog kidney extracts had a mean activity of 0.35 U/g, as measured by stimulation of hemoglobin synthesis in cultured bone marrow cells (in vitro assay) and produced a dose-dependent stimulation of 59Fe incorporation into circulating red cells when assayed in polycythemic mice. Extracts of rabbit kidney cortices had a mean activity of 2.12 U/g, as measured by stimulation of hemoglobin synthesis in cultured bone marrow cells. When the dog kidney homogenate was fractionated on DEAE-cellulose, all of the erythropoietin activity was adsorbed to the exchanger in the presence of 0.01 M acetate buffer, pH 4.5, and was completely eluted by 0.1 M Na2HPO4-0.5 M NaCl, pH 8. An antibody made against human urinary erythropoietin completely inactivated the erythropoietic factor in the dog kidney extract. Serum from a donor dog had no erythropoietin activity when assayed in the starved rat, suggesting that the factor in the extracts is intracellular erythropoietin rather than that contained in plasma trapped in the renal vasculature. The complete inactivation of the erythropoietic factor in these kidney homogenates by antierythropoietin and its behavior on DEAE-cellulose indicate that this factor is structurally similar to native plasma erythropoietin. The extracts are completely active without being incubated in the presence of serum.


The irreversible inactivation of thyroid peroxidase by methylmercaptoimidazole, thiouracil, and propylthiouracil in vitro and its relationship to in vivo findings. A reinvestigation of the mechanism of action of methylmercaptoimidazole, propylthiouracil, and thiouracil on thyroid peroxidase (TPO) was undertaken. A preliminary incubation of TPO and H2O2 with methylmercaptoimidazole, propylthiouracil, or thiouracil was carried out in the absence of oxidizable substrates (i.e. I- or guaiacol). This incubation resulted in irreversible inactivation of TPO. The extent of inactivation could be determined after removal of the drug by gel filtration or by dilution into the assay mixture. Preincubation, as above, in the presence of iodide or thiocyanate prevented the irreversible inactivation of TPO. Rats receiving doses of these drugs which completely inhibited protein-bound iodine formation showed normal levels of TPO in their thyroid glands 30 min after drug administration. These findings suggest that the initial in vivo action of these drugs is to block iodination by trapping oxidized iodide, not by acting as "general inhibitors" of the TPO.