Growth after recombinant human growth hormone treatment in children with chronic renal failure: report of a multicenter randomized double-blind placebo-controlled study. Genentech Cooperative Study Group.

OBJECTIVE: To determine whether treatment with recombinant human growth hormone (rhGH) enhances growth rate in growth-retarded children with chronic renal failure. DESIGN: One hundred twenty-five prepubertal growth-retarded children with chronic renal failure were randomly assigned to receive either rhGH (n = 82) or placebo (n = 43) for 2 years. SETTING: The study was undertaken at 17 pediatric nephrology centers in the United States. MEASUREMENTS: Growth rate, standardized height, bone age, fasting and 2-hour postprandial glucose and insulin levels, biochemical values, and insulin-like growth factor I and anti-growth hormone antibody levels were evaluated serially during the 2-year study period. RESULTS: Standardized height increased from -2.94 to -1.55 in the rhGH group after 24 months of treatment, and decreased from -2.82 to -2.91 in the placebo group (p < 0.00005). Patients in the rhGH group who completed 24 months of study (n = 55) had a greater growth rate during the first year (10.7 +/- 3.1 cm/yr) than during the second year (7.8 +/- 2.1 cm/yr) of treatment. These growth rates were significantly greater than those in the placebo group (n = 27) in the first (6.5 +/- 2.6 cm/yr) and second (5.5 +/- 1.9 cm/yr) years (p < 0.00005 for both years). The mean delta height age minus the delta bone age was positive in the rhGH group, suggesting improved final height potential. There was no significant difference in the change in calculated creatinine clearance over baseline values in the rhGH group from that in the placebo group after 24 months of study (p = 0.63). Mean fasting and postprandial insulin values were elevated at 12 months but not at 24 months in the rhGH-treated patients. Mean fasting and 2-hour postprandial glucose values at 24 months were not significantly elevated over baseline values in either group. CONCLUSIONS: Growth rate and standardized height were significantly increased in growth-retarded prepubertal children with chronic renal failure when rhGH was used. The acceleration in growth was not associated with undue advancement of bone age. No clinically significant side effects were associated with rhGH treatment. The use of rhGH in growth-retarded children with chronic renal failure may facilitate the achievement of the inherent growth potential of such children.

Growth factor modulation of insulin-like growth factor-binding proteins in rat osteoblast-like cells.

Insulin-like growth factors (IGFs) stimulate growth and differentiation of osteoblasts in culture, and these biological functions can be modulated by their binding proteins (IGFBPs). Previous studies have shown that IGFBP-2 is the major IGFBP synthesized by fetal rat osteoblast-like (ROB) cells, which also secret a minor 24-kilodalton IGFBP, presumably IGFBP-4. In this study we examined the modulation of the abundance of IGFBPs by various growth factors. By immunoprecipitation and ligand blotting, we have proved that the 24 kilodalton protein, which appeared at 25 kilodalton in the present study, is IGFBP-4, whose level was strongly enhanced by basic fibroblast growth factor (bFGF) and platelet-derived growth factor BB homodimer. Induction of IGFBP-4 by these factors was detected at 1 nM (10- to 30-fold) and increased to 50- to 70-fold of control at 10 nM. The abundance of IGFBP-4 was also increased but to a lesser degree by transforming growth factor-alpha (TGF-alpha) and epidermal growth factor (EGF). As opposed to the actions of other growth factors, TGF-beta 1 substantially lowered the levels of IGFBP-2 and IGFBP-4. PTH and PTH-related peptide did not induce IGFBPs in ROB cells. This is in contrast to the findings from a malignant rat osteoblast-like cell line, UMR 106-01. Since the level of IGFBP-4 after bFGF treatment remained elevated in the presence of hydroxyurea, the induction was likely to be independent of the stimulatory effects of these factors on mitogenesis. To further examine the mechanisms by which IGFBPs were regulated, cultures were treated with actinomycin D and cycloheximide with and without bFGF. Although the synthesis of IGFBP-2 and IGFBP-4 were both inhibited by cycloheximide, actinomycin D blocked the synthesis of basal and bFGF-induced IGFBP-4 but not IGFBP-2. Total RNA extracted from ROB cells and hybridized with specific rat complementary DNAs for IGFBP-2 and IGFBP-4 showed single transcripts of 1.3 and 2.2 kilobases, respectively. Regardless of the changes at the protein level, the abundance of IGFBP-4 transcripts was not different from the vehicle-treated controls at 2, 8, and 24 h after bFGF treatment. Similarly, the levels of messenger RNA for IGFBP-2 and IGFBP-4 did not change during the same time course in the TGF-beta 1 treatment. These data demonstrate that in ROB cells, the abundance of IGFBPs is differentially regulated by various growth factors.(ABSTRACT TRUNCATED AT 400 WORDS)

A low dose euglycemic infusion of recombinant human insulin-like growth factor I rapidly suppresses fasting-enhanced pulsatile growth hormone secretion in humans.

To determine if insulin-like growth factor I (IGF-I) inhibits pulsatile growth hormone (GH) secretion in man, recombinant human IGF-I (rhIGF-I) was infused for 6 h at 10 micrograms.kg-1.h-1 during a euglycemic clamp in 10 normal men who were fasted for 32 h to enhance GH secretion. Saline alone was infused during an otherwise identical second admission as a control. As a result of rhIGF-I infusion, total and free IGF-I concentrations increased three- and fourfold, respectively. Mean GH concentrations fell from 6.3 +/- 1.6 to 0.59 +/- 0.07 micrograms/liter after 120 min. GH secretion rates, calculated by a deconvolution algorithm, decreased with a t 1/2 of 16.6 min and remained suppressed thereafter. Suppression of GH secretion rates occurred within 60 min when total and free IGF-I concentrations were 1.6-fold and 2-fold above baseline levels, respectively, and while glucose infusion rates were < 1 mumol.kg-1.min-1. During saline infusion, GH secretion rates remained elevated. Infusion of rhIGF-I decreased the mass of GH secreted per pulse by 84% (P < 0.01) and the number of detectable GH secretory pulses by 32% (P < 0.05). Plasma insulin and glucagon decreased to nearly undetectable levels after 60 min of rhIGF-I. Serum free fatty acids, beta-hydroxybutyrate, and acetoacetate were unaffected during the first 3 h of rhIGF-I but decreased thereafter to 52, 32, and 50% of levels observed during saline. We conclude that fasting-enhanced GH secretion is rapidly suppressed by a low-dose euglycemic infusion of rhIGF-I. This effect of rhIGF-I is likely mediated through IGF-I receptors independently of its insulin-like metabolic actions.

The pharmacokinetics of recombinant human relaxin in nonpregnant women after intravenous, intravaginal, and intracervical administration.

The pharmacokinetics of recombinant human relaxin (rhRlx) after intravenous (iv) bolus administration and the absorption of rhRlx after intracervical or intravaginal administration were determined in nonpregnant women. The study was conducted in two parts. In part I, 25 women received 0.01 mg/kg rhRlx iv. After a minimum 7-day washout period, these women were dosed intracervically (n = 10) or intravaginally (n = 15) with 0.75 or 1.5 mg rhRlx, respectively, in 3% methylcellulose gel. Part II was a double-blind, randomized, three-way crossover study in 26 women. At 1-month intervals, each woman received one of three intravaginal treatments consisting of 0 (placebo), 1, or 6 mg rhRlx in 3% methylcellulose gel. The serum concentrations of relaxin following iv administration were described as the sum of three exponentials. The mean (+/- SD) initial, intermediate, and terminal half-lives were 0.09 +/- 0.04, 0.72 +/- 0.11, and 4.6 +/- 1.2 hr, respectively. Most of the area under the curve was associated with the intermediate half-life. The weight-normalized clearance was 170 +/- 50 mL/hr/kg. The observed peak concentration was 98 +/- 29 ng/mL, and the weight-normalized initial volume of distribution was 78 +/- 40 mL/kg, which is approximately equivalent to the serum volume. If central compartment elimination was assumed, the volume of distribution at steady state (Vss/W) was 280 +/- 100 mL/kg, which is approximately equivalent to extracellular fluid volume. Vss/W could be as large as 1300 +/- 400 mL/kg without this assumption.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of recombinant human insulin-like growth factor-I (rhIGF-I) on total and free IGF-I concentrations, IGF-binding proteins, and glycemic response in humans.

To examine the effects of repeated administration of recombinant human insulin-like growth factor-I (rhIGF-I) on IGF-I levels, free IGF-I pharmacokinetics, glycemic response, and IGF-binding proteins (IGFBP), we administered rhIGF-I (0.03 mg/kg iv bolus) to 12 healthy males each morning for 5 consecutive days. Serum was collected over 24 h on days 1 and 5 for measurement of total and free IGF-I, glucose, insulin, and IGFBP. Total IGF-I was measured by RIA after acid/ethanol extraction. Free IGF-I was separated from binding protein-complexed IGF-I using size exclusion high performance liquid chromatography before measurement by RIA. IGFBP were quantitated by optical densitometry of Western ligand blots. Total IGF-I increased significantly from 0-24 h after administration on day 1 (mean +/- SD, micrograms/L: 120 +/- 44 to 166 +/- 51, P = 0.0002) but did not increase significantly from 24 h on day 1 to 0 h on day 5 (166 +/- 51 to 178 +/- 62) or from 0-24 h on day 5 (178 +/- 62 to 209 +/- 89). The area under the total IGF-I concentration curve was greater on day 5 than day 1 (311 +/- 99 min.g/L vs. 249 +/- 77, P = 0.0001). There were no significant differences in free IGF-I concentration or pharmacokinetic parameters or in the degree or timing of hypoglycemia between days 1 and 5. Plasma insulin levels decreased significantly following rhIGF-I administration (day 1 baseline: 53 +/- 11 pmol/L, nadir: 18 +/- 6 pmol/L at 30 min, P = 0.003); day 5 baseline: 47 +/- 15 pmol/L, nadir: 16 +/- 8 pmol/L at 30 min, P = 0.0003. Western ligand blotting revealed the transient appearance of a 30-kilodalton band which migrates in a manner similar to IGFBP-1. This band was undetectable at baseline, peaked between 150 and 210 min after rhIGF-I administration, and diminished by 480-600 min. The response was similar on days 1 and 5. There were no substantial changes in the serum levels of any other IGFBP. In summary, repeated iv bolus administration of rhIGF-I increased the level of total circulating IGF-I without changing free IGF-I disposition or glycemic response. A 30-kilodalton IGFBP band, most likely IGFBP-1, appeared transiently following rhIGF-I administration, probably as a result of suppression of insulin levels. IGFBP-2, -3, and -4 were unaffected.

The role of development and adrenal steroids in the regulation of the mineralocorticoid receptor messenger RNA.

The ontogeny, adrenal-feedback regulation and regional distribution of the mineralocorticoid receptor (MR) mRNA were examined in the rat brain and kidney. In the kidney, MR mRNA levels in the adult were only 25-30% of the neonatal concentration. Adrenalectomy caused a 35% increase in total brain MR mRNA and a 94% increase in kidney MR mRNA levels. Examination of the regional distribution of the MR mRNA within the brain revealed that the hippocampus had the highest levels, and the mRNA abundance increased after adrenalectomy. The administration of dexamethasone to intact animals resulted in a significant reduction of MR mRNA in the kidney of neonatal rats but not in the brain. These data indicate that there are developmental changes in MR gene expression in kidney and that adrenal steroids can modulate MR gene expression in both the brain and kidney.

Ontogeny of angiotensinogen mRNA and angiotensin II receptors in rat brain and liver.

The renin-angiotensin-system (RAS) is active in fetal and neonatal life. This study was undertaken to examine the ontogenic regulation of angiotensinogen (AT) gene expression and angiotensin II (A II) receptors in liver and brain. AT gene expression was studied in fetal, neonatal, adult and aged rats, using slot blot hybridization to quantify AT mRNA levels. During fetal life (gestational days 15-20), AT mRNA was more abundant in brain than in liver. Soon after birth, brain AT mRNA levels increased to a concentration 3 fold above fetal levels. In contrast, liver AT mRNA abundance increased 30-fold within 12 h of birth. Aging (3-20 months) resulted in a gradual decrease in AT mRNA in both the brain and liver. Liver A II receptors in the neonate were 2-fold higher than in the fetus, but returned to fetal levels by 8 weeks of age. In the brain, A II receptor abundance increased to a level 75% above fetal levels in 7 days old animals, but returned to fetal levels by 14 days of age. These studies suggest than in the fetus, the liver is not the primary source of AT but that unknown factors at parturition result in a dramatic increase in liver AT mRNA. In contrast, the more modest increases in brain AT mRNA parallel the gradual maturation of the CNS. In both tissues, further aging resulted in a gradual decrease in AT mRNA, reflecting either increased sensitivity to feedback downregulation by A II or age related increases in other extrahepatic sites of AT synthesis. Age related changes were also found in the A II receptor in both the liver and brain.

Dexamethasone and 1,25-dihydroxyvitamin D3 modulation of insulin-like growth factor-binding proteins in rat osteoblast-like cell cultures.

Using the method of Western ligand blot, we have found that the major form of insulin-like growth factor-binding protein (IGF-BP) secreted by rat osteoblastic-like cells in culture is a 31-kDa protein that is immunologically identical to BP-2, the binding protein originally identified in conditioned medium of Buffalo rat liver cells (BRL-3A). Two minor forms of IGF-BPs with apparent mol wt of 24 kDa (BP-24) and 42 kDa (BP-42) have also been identified. The amount of IGF-BPs in serum-free conditioned medium increased 3-fold on day 3 compared to the day 1 level. We also studied the modulation of IGF-BPs by dexamethasone (DEX), 1,25-dihydroxyvitamin D [1,25-(OH)2D3], and insulin-like growth factor-I (IGF-I). DEX coordinately reduced the level of IGF-BPs in a dose- and time-dependent manner, which resulted in less than 10% of the BP-2 remaining at 100 nM. In contrast, 1,25-(OH)2D3 at 100 nM enhanced the amount of BP-2 by 1-fold. In combined treatments, 1,25-(OH)2D3 at 10 nM was unable to antagonize the inhibitory effect of DEX in the dose range of 1-10 nM. IGF-I, at 1 and 10 nM, proved to be a potent stimulator of all IGF-BPs, and at 10 nM, it completely reversed the inhibition by 100 nM DEX. Although the roles of IGF-BPs have not been clearly defined in bone cells, they are capable of modulating the biological actions of IGFs in other cell culture systems. Modulation of the IGF-BP level by DEX, 1,25-(OH)2D3, and IGF-I suggests important roles for these binding proteins in altering IGF-I action in rat osteoblast-like cell cultures.

Developmental and hormonal regulation of glucocorticoid receptor messenger RNA in the rat.

The biological action of glucocorticoids is dependent upon tissue-specific levels of the glucocorticoid receptor (GR). During stress, the hypothalamic-pituitary-adrenal axis is stimulated, and high levels of glucocorticoids circulate. This axis is modulated by negative feedback by glucocorticoids, which inhibit hypothalamic and pituitary hormone secretion and downregulate GR gene expression. To study the developmental tissue-specific regulation of the GR, we measured the relative concentration of GR mRNA in fetal, neonatal, adult, and aged rats and examined the effects of dexamethasone on GR gene expression. Three different tissue-specific developmental patterns of GR mRNA accumulation were found. In addition, there was an age-dependent tissue-specific pattern in the feedback regulation of GR mRNA by glucocorticoids. In the fetus and neonate, GR mRNA abundance was not regulated by circulating glucocorticoids. The adult pattern of glucocorticoid feedback inhibition of GR mRNA expression appeared between 2 and 7 d of life in liver, and after 7 but before 14 d of age in brain. The GR was biologically active in the 2-d-old neonate, however, since dexamethasone enhanced gene expression of angiotensinogen, which is another glucocorticoid responsive gene. These data demonstrate that the GR gene is regulated by both developmental and tissue-specific factors, and provide another molecular basis for ontogenic variations in the hypothalamic-pituitary-adrenal response to stress.

Development of more potent atrial natriuretic factor (ANF) analogs.

Four modified atrial natriuretic factor (ANF) analogs were designed and synthesized by the solid-phase method. Using human ANF-(99-126) as the reference compound the receptor binding affinity and biological activity of these analogs were examined by radioreceptor assay and in vivo experiments. PLO68, a 21 amino peptide with structural modifications des-Ser103,104-[Mpr105,D-Ala107,114]APII-amide exhibited 2.5 and 2.2 fold more activity than human ANF-(99-126) in lowering blood pressure and causing natriuresis in urethane anesthetized rats. Receptor binding assays using rat lung membranes showed that PLO68 had a Kd of 200 +/- 12 pM compared to a Kd of 620 +/- 12 pM for human ANF-(99-126). Similar chemical modifications, except for substitution of glycine and alanine at the positions 115 and 118, and 120 by D-alanine resulted in three analogs PLO63 and PLO64, PLO67 respectively. PLO63, PLO64 and PLO69 had similar affinities and in vivo potency as human ANF-(99-126). These data suggest that the structural modifications made in PLO68 can cause an increase in the receptor binding ability and an enhancement of biological activities.

Angiotensinogen mRNA. Regulation by cell cycle and growth factors.

The components of the renin-angiotensin system have been colocalized in many tissues suggesting that local generation of angiotensin II can regulate blood flow in specific organs or tissues. This in combination with the fact that proliferating tissues require angiogenesis and increased blood flow to develop have led us to study the relationship of angiotensinogen mRNA production to cell cycle regulation. Reuber H35 (H4IIE) cells were growth-arrested by serum deprivation. Cells were then treated with 10% fetal calf serum, depleted serum, or insulin. Insulin and serum were equally potent at increasing beta-actin mRNA levels, depressing angiotensinogen mRNA levels, and in increasing [3H]methyl thymidine incorporation. The half-maximal insulin effect occurred at 5 x 10(-9) M. Insulin-like growth factor I and II had no effect on any of the parameters measured. 12-O-tetradecanoyl phorbol 13-acetate (TPA) also induced beta-actin mRNA, decreased angiotensinogen mRNA, and caused an increase in [3H]methyl thymidine incorporation. The TPA effects were of shorter duration and of lower magnitude than those caused by insulin or serum. Inactivation of protein kinase C by preincubation with TPA did not block the insulin response. TPA has been shown to induce angiogenesis in vitro. Thus, these studies suggest that inhibition of angiotensinogen gene activity may be part of the proliferative or angiogenic process. Our experimental data may provide a model for further experimental dissection of the biochemical steps involved in angiogenesis.

Multiple hormones regulate angiotensinogen messenger ribonucleic acid levels in a rat hepatoma cell line.

Angiotensinogen is regulated by both hormones and changes in cardiovascular and electrolyte status. We have used the Reuber H35 (H4IIE) rat hepatoma cell line to study the regulation of angiotensinogen mRNA levels by dexamethasone, aldosterone, L-T3, and 17 beta-estradiol. Using the Acc I (1097 basepairs) fragment of our angiotensinogen cDNA clone, we have studied, by Northern and slot blot analysis, the accumulation of angiotensinogen mRNA in this cell culture system. Angiotensinogen mRNA of approximately 1800 bases was identified in these cells. It was identical in size to angiotensinogen mRNA from rat liver. Cells grown in medium containing serum depleted of thyroid and steroid hormones for up to 72 h showed a progressive decrease in angiotensinogen mRNA. Dexamethasone treatment resulted in a time- and dose-dependent increase in angiotensinogen mRNA. The half-maximal response occurred at 10(-9) M dexamethasone, with a maximal response of approximately 4-fold (serum-free conditions). Aldosterone induced a dose-dependent increase in mRNA. Half-maximal levels were obtained at 5 X 10(-8) M. Competition studies using the glucocorticoid antagonist RU38486 (Roussel-UCLAF) confirmed that dexamethasone was acting through the glucocorticoid receptor and suggested that aldosterone was acting through the same receptor. L-T3 treatment caused a dose and time-dependent increase in angiotensinogen mRNA levels. The half-maximal response occurred at 5 X 10(-8) M, and the maximal response was a 2-fold increase. Combined treatment with dexamethasone and L-T3 triiodothyronine resulted in a synergistic increase in angiotensinogen mRNA levels. 17 beta-Estradiol failed to elicit a change in angiotensinogen mRNA levels consistent with the observation that these cells lack estrogen receptors. Our results indicate that hepatic angiotensinogen mRNA levels are regulated in a complex fashion by several hormones. These cells provide a useful system for studying the hormonal regulation of the angiotensinogen gene.

Tissue-specific regulation of glucocorticoid receptor mRNA by dexamethasone.

The effect of glucocorticoids on tissue-specific regulation of glucocorticoid receptor mRNA was studied in intact and adrenalectomized rats. Glucocorticoid receptor mRNA was examined by Northern blot hybridization and quantitated by slot blot hybridization using a glucocorticoid cRNA probe. Glucocorticoid receptor mRNA was greatest in the lung with the relative levels in other tissues as follows: spleen, 70%; brain, 55%; liver, 50%; kidney, 43%; heart, 35%; adrenal, 13%; and testis only 8%. A tissue-specific difference in glucocorticoid receptor mRNA accumulation was found after adrenalectomy. There was little change in glucocorticoid receptor mRNA levels in liver and lung, but the brain and kidney demonstrated a 40 and 80% increase in mRNA, respectively. In contrast, dexamethasone treatment resulted in a consistent decrease of 40-60% in the accumulation of glucocorticoid receptor mRNA in all tissues studied. These results provide in vivo evidence for the autoregulation of the glucocorticoid receptor by its homologous ligand and demonstrate the existence of tissue-specific regulation of the glucocorticoid receptor mRNA levels in states of glucocorticoid excess and depletion.

Tissue-specific regulation of angiotensinogen mRNA accumulation by dexamethasone.

The regulation of angiotensinogen gene expression in response to adrenalectomy and dexamethasone treatment was examined in multiple rat tissues. Angiotensinogen mRNA as quantitated by slot blot hybridization utilizing an angiotensinogen cRNA probe was most abundant in the liver with levels in the brain, kidney, and adrenal of 50, 25, and 10%, respectively. No angiotensinogen mRNA was detected in testes or heart. Although no change in the quantity of angiotensinogen mRNA was found following adrenalectomy and maintenance on 0.9% saline, dexamethasone treatment of both normal and adrenalectomized rats resulted in a time-dependent and tissue-specific accumulation of angiotensinogen mRNA. In normal animals, the hepatic response to treatment was a 4.5-fold increase in angiotensinogen mRNA by 8 h which remained 2.4-fold above basal levels by 24 h. Angiotensinogen mRNA levels in the brains of normal rats treated with dexamethasone increased only 60% by 6 h and returned to basal levels by 24 h. In contrast to the increases seen in brain and liver, angiotensinogen mRNA derived from kidney did not significantly change following dexamethasone treatment. In adrenalectomized animals, the hepatic response to dexamethasone was similar to normal animals with a 3.7-fold increase by 6 h. The accumulation in brain was greater in these animals compared to normals and increased 3-fold by 8 h. Finally, dexamethasone did not significantly increase levels in the kidney. These results clearly demonstrate glucocorticoid regulation of angiotensinogen mRNA levels in liver and brain. In contrast, the kidney, an organ known to contain glucocorticoid receptors, does not respond with increased angiotensinogen mRNA levels following glucocorticoid stimulation. These studies provide the first evidence for tissue-specific differences in the control of angiotensinogen mRNA.

Transient paralysis by heat shock of hormonal regulation of gene expression.

We have investigated the effect of heat shock on primary cultures of male and female Xenopus laevis hepatocytes as a function of estrogen-induced vitellogenin gene expression. Coincident with the induction of heat-shock protein (hsp) synthesis, thermal stress abolishes the estrogen activated transcription and accumulation of vitellogenin mRNA, at the same time causing the destabilization of vitellogenin mRNA accumulated by prior treatment with the hormone. Exposure of the cells to estrogen before heat shock allows an immediate resumption of vitellogenin gene transcription on return to 26 degrees C. Heat shock applied to cells from hormonally naive male Xenopus extends the lag period preceding vitellogenin gene transcription upon return to normal temperatures. This transient and reversible paralysis of estrogen responsiveness is paralleled by reversible changes in the amount of nuclear estrogen receptor in the hepatocytes. Heat shock therefore offers a novel approach in the manipulation and analysis of the early stages of steroid hormonal regulation of gene expression.

Regulation by estrogen receptor of vitellogenin gene transcription in Xenopus hepatocyte cultures.

We have used primary cell cultures of hepatocytes from male or female Xenopus laevis to study the mechanisms by which estrogen induces vitellogenin gene transcription and how primary exposure to estrogen renders cells more responsive to secondary stimulation. We have characterized the estrogen receptor in hormonally naïve cells and in hepatocytes treated with estrogen under a variety of conditions. Under all conditions the receptor has a Kd congruent to 4 X 10(-10) M. Hormonally naïve male cells contain 300 binding sites whereas female cells or male cells previously exposed to estradiol exhibit 6-7-fold higher levels. In parallel cultures, the absolute rate of vitellogenin gene transcription was determined by hybridization of newly synthesized RNA pulse-labelled with [3H]uridine to cloned Xenopus vitellogenin cDNA. Naïve male cells on primary stimulation with estradiol synthesized vitellogenin mRNA at an average rate of approximately 150 moles/cell/h compared to 1200 moles/cell/h for cells previously exposed to estrogen, thus bearing a close correlation with receptor number. Furthermore, we show that the kinetics of the induced up-regulation of receptor exactly parallel those of the increase in the rate of vitellogenin gene transcription upon secondary hormonal stimulation following various periods of primary exposure to estrogen. Addition of cycloheximide to cell cultures during primary estrogen treatment abolishes both receptor up-regulation and increased rate of vitellogenin gene transcription on secondary stimulation. In addition, primary treatment with the antiestrogen tamoxifen prevents both receptor up-regulation and an enhanced rate of transcription or accumulation of vitellogenin mRNA on secondary hormonal exposure. These results demonstrate that estrogen treatment of male Xenopus hepatocytes results in the rapid up-regulation of its own receptor to female levels via new receptor synthesis, and that receptor number is rate-limiting in vitellogenin gene transcription.

Central and peripheral control of siphon-withdrawal reflex in Aplysia californica.

1. The defensive withdrawal reflex of the siphon of Aplysia is a local response (exhibited by the organ that is stimulated) mediated by the conjoint action of both the central and peripheral nervous systems. 2. Three independent methods were used to determine the contribution of the central and peripheral nervous systems to the siphon-withdrawal reflex: 1) acute reversible deganglionation, 2) chronic deganglionation, and 3) a selective reversible hyperpolarization. With each of these techniques, the central nervous system was found to contribute about 55% of the total reflex. 3. Seven motor neurons were identified and characterized with respect to their electrophysiological properties and the motor actions. Three of the central motor cells belong to the LD clusters of cells (LDS1, LDS2, LDS3) and one is an RD cell (RDS). These four cells all receive excitatory synaptic input from siphon stimulation, excitatory synaptic input from the activity of the respiratory command cells network (interneuron II). large spontaneous IPSPs, and exhibit hyperpolarizing responses (H response) to iontophoretically applied acetylcholine (ACh). These cells all participate in the siphon-withdrawal component of a centrally commanded fixed-action pattern: spontaneous pumping movements of the mantle organs driven by the respiratory command cells. They receive an EPSP burst during the activity of the respiratory command cells and are competent to mediate the siphon motion. Three central siphon motor cells belong to the LB cluster (LBS1, LBS2, LBS3). These cells also receive excitatory input following stimulation of the siphon, a spontaneously occurring IPSP, and have H response to iontophoretically applied ACh. These cells, however, receive an IPSP burst during spontaneous pumping movement and thus do not participate in the active contraction phase of this behavior. LBS1 and LDS1 were examined with respect to their transmitter biochemistry and were found to be noncholinergic. 4. The siphon-withdrawal reflex habituates with comparable kinetics to repeated tactile stimulation when it is under central and peripheral control and when it is under peripheral control only. Thus, not only do both systems act conjointly to produce the defensive withdrawal reflex, but also they have similar response properties and are well matched to mediate the two parts of this siphon behavior.