Thyroid hormone: Aldosterone antagonism in cultured epithelial cells

Thyroid hormone (T₃) has been demonstrated to inhibit the action of aldosterone on sodium transport in toad urinary bladder and rat kidney. We have exammined the effect of T₃ on aldosterone action and specific nuclear binding in cultured epithelial cells derived from toad urinary bladder. In cell line TB6-C, addition of 5·10⁻⁸ M T₃ to culture media for up to 3 days results in no change in short-circuit current or transepithelial resistance. This concentration of T₃ completely inhibits the maximal increase in short-circuit current in response to 1·10⁻⁷ M aldosterone. The inhibition can be demonstrated with 18 h preincubation or with simultaneous addition of T₃ and aldosterone. The half-maximal concentration for the inhibition of the aldosterone effect is approx. 5·10⁻⁹ M T₃. T₃ has no effect on cyclic AMP-stimulated short-circuit current in these cells. The effect of T₃ on nuclear binding of [³H]aldosterone was examined using a filtration assay with data analysis by at least-squares curve-fitting program. Best fit was obtained with a model for two binding sites. The dissociation constants for the binding were K′_d1 = (0.82 ± 0.36)·10⁻¹⁰ M and K′_d2 = (3.2±0.60)·10⁻⁸ M.The half-maximal concentration for aldosterone-stimulated sodium transport in these cells is approx. 1·10⁻⁸ M. Analysis of nuclear aldosterone binding in cells preincubated for 18 h with 5·10⁻⁸ M T₃ showed a K′_d1 = (0.15 ± 0.10)·10⁻¹⁰ M and K′_d2 = (3.5 ± 0.10)·10⁻⁸ M. We conclude that T₃ i action of aldosterone on sodium transport at a site after receptor binding in the nucleus.     

Aldosterone-stimulated sodium uptake by apical membrane vesicles from A6 cells

Sodium fluxes in plasma membrane vesicles prepared from the cultured toad kidney epithelial cell line A6 are studied. The vesicles are enriched 7-10 times in apical membrane markers. Sodium uptake is osmotically sensitive and inhibited by low concentrations of amiloride (K0.5 = 7 X 10(-8) M at 1 mM NaCl). Vesicles prepared from aldosterone-treated cells (4.5 h at 10(-7) M aldosterone) show a 2-fold enhancement of amiloride-sensitive sodium flux relative to appropriate controls. The above observations are in good agreement with studies of sodium transport across the apical membrane of intact A6 epithelia. Thus, the amiloride-sensitive sodium transporter in the apical membrane of these cells is preserved in the vesicle preparation, making it possible to study the effects of aldosterone in the absence of nonmembrane-related phenomena.     

Spironolactone antagonism of aldosterone action on Na+ transport and RNA metabolism in toad bladder epithelium.

In earlier studies, aldosterone increased the incorporation of precursors into a class of cytoplasmic RNA with the characteristics of messenger RNA (mRNA), in toad bladder epithelium. In the present studies, this effect was analyzed further with a competitive antagonist, spironolactone (SC-9420). Paired hemibladders were labeled with 3H-uridine (30 min pulse - 140 min chase), with or without aldosterone (3.5 x 10(-8) M, 7 X 10(-8) M) in the presence or absence of SC-9420 (7 X 10(-6) M, 2.5 X 10(-5) M) at molar ratios of 200:1 to 280:1. Cytoplasmic RNA, either the total phenol-SDS extract or polyadenylated-RNA (poly(A)(+)-RNA) obtained by oligo-deoxythymidylate-cellulose (oligo(dT)-cellulose) chromatography was analyzed in linear 5 -- 20% sucrose gradients. Eight sets of experiments were completed in which the short-circuit current (scc) was monitored for 180 min and the incorporation of 3H-uridine (30 min pulse -- 150 min chase) was simultaneously determined on pools of epithelia from 5 to 10 hemibladders. The fractional change in scc correlated linearly with the fractional change in 3H-uridine of 12S cytoplasmic RNA (r=0.95, p less than 0.001). The poly(A)(+)-RNA fraction had no detectable rRNA or tRNA and gave a heterogeneous pattern, typical of mRNA, in the sucrose gradients. In the presence of exogenous aldosterone, SC-9420 inhibited the incorporation of 3H-uridine into poly(A)(+)-RNA (particularly 12S). These results support the inference that induction of mRNA mediates the action of aldosterone on Na+ transport.     

Thyroid hormone receptors in human cultured fibroblasts: evidence for cellular T4 transport and nuclear T3 binding

In cultured normal human skin fibroblasts specific and saturable binding sites for triiodothyronine (T3) have been revealed. In fact radiolabelled T3 binds rapidly to intact cells with maximum uptake after 1 hour, while nuclear binding is delayed, the equilibrium being reached after 2 hours. In intact cells it is possible to identify a single binding site for 125I-T3, with a Ka = 1.8 X 10(10)M-1 and Ro = 1.25 X 10(-11)M, similarly in nuclei it was possible to identify a single binding site of Ka = 8.8 X 10(9)M-1 and Ro = 2.3 X 10(-11)M. Intact human fibroblasts take up thyroxine (T4) even more rapidly than T3, with maximum after 5 min, showing a lower affinity for T4 than for T3 and a negligible specific and saturable binding sites for T4, the presence of a cellular transport system for T4 may be hypothesized, considering that iodothyronine cellular binding is increased by preincubation with low doses of T4.     

Aldosterone nuclear receptors in kidneys of chick embryo

We have studied the properties of the nuclear receptors for aldosterone in kidneys of chick embryo. Aliquots of 0.4 M KCl nuclear extracts were incubated with [3H]aldosterone with or without 1 microM RU28362, a potent glucocorticoid analog. Scatchard analyses of binding data revealed two classes of binding sites with Ka of 0.26 and 0.03 X 10(9) M-1 and Nmax of 330 fmol and 620 fmol/mg DNA respectively. In presence of RU28362, however, we observed only a single class of binding sites with a Ka of 1.02 X 10(8) M-1 and a Nmax of 90 fmol/mg DNA. Competition studies performed in presence of RU28362 showed that aldosterone was the more effective competitor followed by corticosterone, progesterone, deoxycorticosterone, dexamethasone, cortisol, triamcinolone acetonide and cortisone. The nuclear complexes had a sedimentation coefficient in the area of 8 S which changed to 4-5 S in the presence of 0.4 M KCl. This effect of KCl was prevented by the addition of 10 mM sodium molybdate. Always in the presence of the glucocorticoid analog, by DEAE-c chromatography we observed a major specific aldosterone-binding fraction which was eluted with 0.2 M KCl. This fraction sedimented at 8.4 S in the absence of sodium molybdate and KCl. In the absence of RU28362, DNA-c columns retained only a small portion of the nuclear complexes which were eluted with KCl. These complexes sedimented, on sucrose gradient, at 4.6 and 3.1 S, whereas those which did not bind to DNA-c had a sedimentation coefficient of 8 S. In the presence of RU28362, the majority of bound [3H]aldosterone remained in the column flow-through fraction; when this fraction was further analyzed on DEAE-c, complexes were eluted with 0.2 and 0.3 M KCl. These data indicate that nuclear receptors for aldosterone are present in small number in kidneys of chick embryo and that they are mostly in the 8 S form.     

Stimulation of sodium transport by aldosterone and arginine vasotocin in A6 cells

The effects of aldosterone and arginine vasotocin (AVT) on transepithelial Na+ transport of cultured A6 cells were investigated. All experiments were performed with cells grown on Millicell TM culture-plate inserts for a period of 2-4 weeks in defined, serum-free medium. Omitting fetal bovine serum 2 days after seeding the cells on filters did not influence potential difference (PD) development or the hormonal responses tested. The cell layers were placed in an Ussing chamber for short-circuit current (ISC) and transepithelial conductance (G) measurements. Base-line values were (n = 93): PD, 51.0 +/- 0.2 mV (apical side negative); ISC, 14.55 +/- 0.06 microA/cm2; G, 0.306 +/- 0.001 mS/cm2. ISC and G were higher in cells pretreated with 10(-7) M aldosterone for 24 h in the incubator, when compared to controls (ISC, 28 +/- 2 vs. 16 +/- 2 microA/cm2, G, 0.41 +/- 0.04 vs. 0.26 +/- 0.01 mS/cm2, n = 5) and both remained stable for at least 6 h. In cells not treated with aldosterone, 10(-7) M AVT increased ISC within 1 min after addition, producing a maximum ISC within 15 min which then declined to baseline levels over the next 5 h. Addition of AVT to aldosterone-pretreated cells resulted in a significantly greater peak increase in ISC than in non-pretreated cells (change in ISC compared to controls: 8.1 +/- 0.4 vs. 4.9 +/- 0.4 microA/cm2, n = 5, P less than 0.001), indicating a synergistic effect. A dose-response curve for amiloride obtained in the presence of AVT showed that amiloride completely inhibits ISC. Pretreatment of the A6 cells with aldosterone for 24 h shifted the amiloride dose-response curve to the right, as expressed in a doubling of the apparent Ki value (from 0.17 +/- 0.02 to 0.33 +/- 0.04 microM). In conclusion, A6 cells grown in defined, serum-free medium express a greater than additive synergism between aldosterone and AVT in stimulating transepithelial Na+ transport.     

Antiestrogen binding sites in rat liver nuclei

Isolated, intact rat liver nuclei have high-affinity (Kd = 10(-9) M) binding sites that are highly specific for nonsteroidal antiestrogens, especially for compounds of the triphenylethylene series. Nuclear [3H]tamoxifen binding capacity is thermolabile, being most stable at 4 degrees C and rapidly lost at 37 degrees C. More [3H]tamoxifen, however, is specifically bound at incubation temperatures of 25 degrees C and 37 degrees C than at 4 degrees C although prewarming nuclei has no effect, suggesting exchange of [3H]tamoxifen for an unidentified endogeneous ligand. Nuclear antiestrogen binding sites are destroyed by trypsin but not by deoxyribonuclease I or ribonuclease A. The nuclear antiestrogen binding protein is not solubilized by 0.6 M potassium chloride, 2 M sodium chloride, 0.6 M sodium thiocyanate, 3 M urea, 20 mM pyridoxal phosphate, 1% (w/v) digitonin or 2% (w/v) sodium cholate but is extractable by sonication, indicating that it is tightly bound within the nucleus. Rat liver nuclear matrix contains high-affinity (Kd = 10(-9) M) [3H]tamoxifen binding sites present in 5-fold higher concentrations (4.18 pmol/mg DNA) than in intact nuclei (0.78 +/- 0.10 (S.D.) pmol/mg DNA). Low-speed rat liver cytosol (20 000 X g, 30 min) contains high-capacity (955 +/- 405 (S.D.) fmol/mg protein), low-affinity (Kd = 10.9 +/- 4.5 (S.D.) nM) antiestrogen binding sites. In contrast, high-speed cytosol (100 000 X g, 60 min) contains low-capacity (46 +/- 15 (S.D.) fmol/mg protein), high-affinity (Kd = 0.61 +/- 0.20 (S.D.) nM) binding sites. Low-affinity cytosolic sites constitute more than 90% of total liver binding sites, high-affinity cytosolic sites 0.3%-3.2%, and nuclear sites less than 0.5% of total sites.     

Binding, surface mobility, internalization, and degradation of rhodamine-labeled alpha 2-macroglobulin

We have used quantitative fluorescence methods to examine the fate of rhodamine-labeled alpha 2-macroglobulin (R-alpha 2 M) after binding to cell-surface receptors on NRK and Swiss 3T3 cells. From measurements of fluorescence intensities in NRK cells fixed after incubation with R-alpha 2M, we found that uptake was saturable and that half-maximal uptake occurred at 130 nM R-alpha 2M. Fluorescence measurements on cell extracts of NRK and Swiss 3T3 cells also showed a half-maximal uptake of R-alpha 2M near 130 nM. We estimate that NRK cells can take up 10(6) molecules of R-alpha 2M per hour via receptor-mediated endocytosis. The mobility of alpha 2-macroglobulin receptors on the surface of Swiss 3T3 cells was measured by using fluorescence photobleaching recovery. The two-dimensional effective diffusion coefficient of R-alpha 2M receptors was approximately 8 X 10(-10) cm2 s-1, a value close to that previously obtained for insulin and epidermal growth factor receptors. Degradation of R-alpha 2M by the cells was followed by using the loss of fluorescence from the 185000-dalton band in sodium dodecyl sulfate--polyacrylamide gels. Rhodamine fluorescence was detected in the gels by using a microscope fluorescence spectrophotometer. NRK cells degraded alpha 2M to low molecular weight fragments with a t 1/2 of 15 min. Swiss 3T3 cells degraded about 75% of the alpha 2M with a t 1/2 of 1 h. The remaining 25% remained as the intact 185000-dalton peptide after 24 h. No significant accumulation of large breakdown products was observed in Swiss 3T3 or NRK cells.     

Proton pump activity is required for active uptake of chloride in isolated amphibian skin exposed to freshwater

Net proton secretion and unidirectional chloride fluxes were measured in isolated skin of toads ( Bufo bufo) and frogs ( Rana esculenta) mounted in an Ussing chamber and exposed to a Ringer's solution on the serosal side and a freshwater-like solution (1-3 mM Cl(-)) on the external side. Active proton secretion was 34.2+/-2.0 pmol.cm(-2).s(-1) ( n=18) in frog skin, and 16.7+/-1.7 pmol.cm(-2).s(-1) ( n=10) in toad skin. Proton secretion by toad skin was dependent on the transepithelial potential ( V(T)), and an amiloride-insensitive short-circuit current was stimulated by exogenous CO(2)/HCO(3)(-), indicating the presence of a rheogenic proton pump. Cl(-) influx was 37.4+/-7.5 pmol.cm(-2).s(-1) ( n=14) in frog skin and 19.5+/-3.5 pmol.cm(-2).s(-1) ( n=11) in toad skin. In toad skin, the mean Cl(-) flux ratio was larger than expected for simple electro-diffusion. In 8 of 11 sets of paired skins, influx was greater than the efflux indicating active uptake of Cl(-). Cl(-) influx in toad skin was unaffected by large perturbations (100-150 mV) of V(T), which was accomplished by adding amiloride to the outer bath under open circuit conditions. A component of the Cl(-) efflux seemed to be dependent on V(T). 4,4'-Diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS; 0.3 mM or 1.3 mM) inhibited Cl(-) influx and, surprisingly, increased Cl(-) efflux in toad skin. Influx and efflux of Cl(-) in toad skin were highly dependent on the external [Cl(-)] in the freshwater range (0.1-4 mM). (36)Cl(-) influx decreased whereas the total Cl(-) efflux increased as a function of external [Cl(-)]. These data indicate the presence of a DIDS-sensitive, electroneutral carrier mechanism with an external binding site for Cl(-). Ethoxzolamide (100 micro M), an inhibitor of carbonic anhydrase, reduced proton secretion and Cl(-) influx in frog skin. Concanamycin A (0.1-10 micro M), a specific vacuolar-type proton pump (V-ATPase) inhibitor, significantly reduced proton secretion in frog skin. In addition, concanamycin A (1 micro M) significantly reduced Cl(-) influx in frog skin. We suggest that the active proton secretion and Cl(-) influx are coupled. We hypothesise that an apical V-ATPase is capable of energising active Cl(-) uptake in fresh water by creating a favourable gradient for an apical HCO(3)(-) exit in exchange for external Cl(-). The data also suggest that a carbonic anhydrase activity provides H(+) and HCO(3)(-) for apically co-expressed proton pumps and Cl(-)/HCO(3)(-) exchangers.     

Bombesin receptor in membranes from Swiss 3T3 cells. Binding characteristics, affinity labelling and modulation by guanine nucleotides.

Bombesin-like neuropeptides, including mammalian gastrin-releasing peptide (GRP), are potent mitogens for Swiss 3T3 cells. In this study, we have characterized the bombesin receptor in membrane preparations from these cells. Addition of Mg2+ during cell homogenization was essential to preserve 125I-GRP binding activity in the resulting membrane preparation. The effect of Mg2+ was concentration dependent, with a maximum at 5 mM. Specific binding of 125I-GRP was saturable; Scatchard analysis indicated a single class of high-affinity sites of Kd = (2.1 +/- 0.3) x 10(-10) M at 15 degrees C and Kd = (1.9 +/- 0.4) x 10(-10) M at 37 degrees C, and a maximum binding capacity of 580 +/- 50 fmol/mg of protein (15 degrees C) or 604 +/- 40 fmol/mg of protein (37 degrees C). The kinetically derived dissociation constant was 1.5 x 10(-10) M. 125I-GRP binding was inhibited in a concentration-dependent manner by various peptides containing the highly conserved C-terminal heptapeptide of the bombesin family, including bombesin, GRP, neuromedin B and the 8-14 fragment of bombesin. In contrast, a variety of structurally unrelated mitogens and neuropeptides had no effect. The cross-linking agent ethyleneglycolbis(succinimidylsuccinate) covalently linked 125I-GRP to a single Mr 75 000-85 000 protein in membrane preparations of 3T3 cells. Affinity labelling of this molecule was specific and dependent on the presence of Mg2+ during membrane preparation. Finally, the non-hydrolysable GTP analogue guanosine-5'-[gamma-thio]triphosphate (GTP[S]) caused a concentration-dependent inhibition of 125I-GRP binding and cross-linking to 3T3 cell membranes [concentration giving half-maximal inhibition (IC50) approximately 0.2 microM]. The inhibitory effect was specific (GMP, ATP or ATP[S] had no effect at 10 microM) and was due to an increase in Kd from (1.7 +/- 0.2) x 10(-10) M to (4.3 +/- 0.6) x 10(-10) M in the presence of 10 microM-GTP[S]. This modulation of ligand affinity and cross-linking implies that the bombesin receptors that mediate mitogenesis in Swiss 3T3 cells are coupled to a guanine-nucleotide-binding-protein signal-transduction pathway.     

Hormonal regulation of prolactin release by human prolactinoma cells cultured in serum-free conditions

Thyrotropin-releasing hormone (TRH) stimulates the prolactin (PRL) release from normal lactotrophs or tumoral cell line GH3. This effect is not observed in many patients with PRL-secreting tumors. We examined in vitro the PRL response to TRH on cultured human PRL-secreting tumor cells (n = 10) maintained on an extracellular matrix in a minimum medium (DME + insulin, transferrin, selenium). Addition of 10(-8) M TRH to 4 X 10(4) cells produced either no stimulation of PRL release (n = 6) or a mild PRL rise of 32 +/- (SE) 11% (n = 4) when measured 1, 2 and 24 h after TRH addition. When tumor cells were preincubated for 24 h with 5 X 10(-11) M bromocriptine, a 47 +/- 4% inhibition of PRL release was obtained. When TRH (10(-8) M) was added, 24 h after bromocriptine, it produced a 85 +/- 25% increase of PRL release (n = 8). This stimulation of PRL release was evident when measured 1 h after TRH addition and persisted for 48 h. The half maximal stimulatory effect of TRH was 2 X 10(-10) M and the maximal effect was achieved at 10(-9) M TRH. When tumor cells were pretreated with various concentrations of triiodothyronine (T3), the PRL release was inhibited by 50% with 5 X 10(-11) M T3 and by 80% with 10(-9) M T3. Successive addition of TRH (10(-8) M) was unable to stimulate PRL release at any concentration of T3. The addition of 10(-8) M estradiol for up to 16 days either stimulated or had no effect upon the PRL basal release according to the cases. In all cases tested (n = 4), preincubation of the tumor cells with estradiol (10(-8) M) modified the inhibition of PRL release induced by bromocriptine with a half-inhibitory concentration displaced from 3 X 10(-11) M (control) to 3 X 10(-10) M (estradiol). These data demonstrate that the absence of TRH effect observed in some human prolactinomas is not linked to the absence of TRH receptor in such tumor cells. TRH responsiveness is always restored in the presence of dopamine (DA) at appropriate concentration. This TRH/DA interaction seems specific while not observed under T3 inhibition of PRL. Furthermore, estrogens, while presenting a variable stimulatory effect upon basal PRL, antagonize the dopaminergic inhibition of PRL release.     

The formylpeptide chemotactic receptor on rabbit peritoneal neutrophils. I. Evidence for two binding sites with different affinities

F Met-Leu-[3H]Phe and f Nle-Leu-[3H]Phe binding to rabbit peritoneal neutrophils and purified membranes were measured at 4 degrees C silicone oil centrifugation assays, and the results were analyzed by the LIGAND computer program, which permits analysis of ligand binding to multiple classes of binding sites. LIGAND analysis of peptide binding to intact neutrophil indicated that both f Met-Leu-[3H]Phe and f Nle-Leu-[3H]Phe detected two population of binding sites. The apparent Kd values for f Met-Leu-[3H]Phe binding were 1.6 +/- 1.0 X 10(-9) M and 2.2 +/- 0.9 X 10(-8) M, respectively, and 3.1 +/- 0.2 X 10(-9) M and 1.2 +/- 0.6 X 10(-7) M for f Nle-Leu-[3H]Phe. Furthermore, the higher affinity sites detected on whole cells comprised approximately 15 to 30% of the total sites. Two populations of binding sites were also detected on purified neutrophil plasma membranes by both radiolabeled chemotactic peptides. LIGAND analysis of peptide binding to purified membranes yielded apparent Kd values of 5.0 +/- 2.5 X 10(-10) M and 4.8 +/- 0.6 X 10(-8) M for f Met-Leu-[3H]Phe binding, and 4.7 +/- 4.2 X 10(-10) M and 3.0 +/- 1.3 X 10(-8) M for f Nle-Leu-[3H]Phe. The percentage of higher affinity sites detected by f Met-Leu-[3H]Phe and f Nle-Leu-[3H]Phe on purified membranes were 1 to 5% of the total sites detected. These data are consistent either with the existence of two independent binding sites for formylpeptides on rabbit neutrophils or receptor negative cooperativity.     

Ca2+-dependent facilitated shortening in isotonic contraction of trachealis muscle

We compared isotonic shortening with isometric force generation as a function of external Ca2+ in 166 tracheal smooth muscle (TSM) strips from 27 mongrel dogs in vitro. Concentration-response curves were generated with muscarinic stimulation (acetylcholine, ACh), alpha-adrenergic receptor activation (norepinephrine after beta-adrenoceptor blockade, NE), serotonin (5-HT), and KCl-substituted Krebs-Henseleit solution. The concentrations of 5-HT causing half-maximal shortening (ECS50, 1.54 +/- 0.14 X 10(-7) M) and half-maximal active isometric tension (ECT50, 1.72 +/- 0.30 X 10(-7) M) were similar (P = NS). Likewise, ECS50 (21.9 +/- 0.7 mM) and ECT50, (22.0 +/- 0.9 mM) were similar for KCl. In contrast, facilitated isotonic shortening (i.e., greater isotonic shortening for comparable degrees of force generation) was elicited with ACh and NE for all levels of force generation between 15 and 85% of maximum and for all concentrations of ACh from 3 X 10(-8) to 3 X 10(-5) M (P less than 0.05 for all points). Facilitated isotonic shortening also was elicited for all concentrations of NE from 10(-8) to 10(-6) M (P less than 0.05 for all points). Removal of Ca2+ from the perfusate substantially reduced the potency of ACh (P less than 0.001) and abolished differences between ECS50 (2.23 +/- 0.28 X 10(-5) M) and ECT50 (2.50 +/- 0.46 X 10(-5) M, P = NS). We demonstrate that for comparable degrees of force generation, muscarinic and alpha-adrenergic receptor activation cause greater isotonic shortening than KCl or 5-HT and that this facilitated shortening is associated with the concentration of external Ca2+.     

Dual effect of vasoactive intestinal peptide on the mitogenic response of rabbit spleen lymphocytes

The effects of vasoactive intestinal peptide (VIP) have been investigated on the mitogenic response of rabbit spleen cells. Specific binding of 125I-VIP to these mononuclear cells is rapid and saturable. Analysis of binding reveals two classes of binding sites, a class of high-affinity binding sites with KD = 0.93 +/- 0.11 nM and maximal binding capacity of 2000 +/- 560 sites/cell, and a class of low-affinity binding sites with KD = 225 +/- 58 nM and maximal binding capacity of 280,000 +/- 60,000 sites/cell. The VIP regulatory effect on mitogen-stimulated rabbit spleen cell proliferation appears to be time dependent and bimodal. When VIP was added simultaneously with mitogens, it induced an inhibition of the proliferative response. With concanavalin A (Con A) or pokeweed mitogen (PWM), addition of 10(-8) M VIP resulted in a maximal 30% inhibition of [3H]thymidine incorporation after 96 h of culture. This inhibitory effect was significant at concentrations from 10(-8)-10(-6) M and half-maximal inhibition was obtained with 1.2 x 10(-9) M VIP. By contrast, when rabbit spleen cells were preincubated for 18 h with VIP alone, the lymphocyte proliferative response to Con A was increased. However, this increase was mitogen-selective, since it was only observed when the T-cell mitogen Con A was used. The maximal response was obtained after 96 h of culture in the presence of Con A. The VIP stimulatory effect was dose-dependent with a maximal effect at 10(-7) M and a half-maximal effect at 1.7 x 10(-9) M VIP. The effect of VIP was also time-dependent, since a 6 h preincubation was sufficient to induce a significant increase in the proliferative response which was maximal after an 18 h preincubation.     

The effects of bumetanide and furosemide on lung liquid secretion in fetal sheep

Thirty-four experiments were carried out on the effects of loop diuretics on lung liquid secretion in 20 fetal sheep (128-145 days gestation) with indwelling catheters. Bumetanide placed in the lung liquid at 2.19 +/- 0.52 X 10(-4) M produced immediate reabsorption of fluid, and effects lasted 3 hr (n = 6). Bumetanide at 1.1 +/- 0.17 X 10(-5) M reduced secretion significantly for 2 hr (n = 4), but at 1.07 +/- 0.06 X 10(-6) M there was no clear effect (n = 6). Controls showed no significant change (n = 6). Furosemide was less effective. At 3.1 +/- 0.07 X 10(-3) M it produced an immediate reabsorption, which lasted 3 hr, but at 1.0 +/- 0.04 X 10(-4) M it increased secretion slightly (n = 4); controls showed no significant change (n = 6). The results are consistent with the presence of a chloride transport system, perhaps with sodium cotransport, as the major factor in fetal lung liquid secretion.     

Androgen receptors in brain and pituitary of female rats: cyclic changes and comparisons with the male

The in vitro binding of a synthetic androgen, methyltrienolone ([3H]-R1881), to brain and pituitary (PIT) cytosol and nuclear extracts was determined in male and female rats. Purified cytosol was prepared from PIT or hypothalamic-preoptic area-amygdala (HPA) and incubated in the presence of 0.1 to 10 nM [3H]-R1881. Scatchard analysis revealed the presence of a single, saturable, high-affinity binding site in PIT cytosol with a dissociation constant (Kd) of 0.42 X 10(-10) M in females and 0.95 X 10(-10) M in intact males. The Kd of HPA cytosol was much less in castrated males [0.47 +/- 0.05 (SEM) X 10(-10)M, n = 7] and females (0.63 +/- 0.1 X 10(-10) M, n = 4) than in intact males (5.8 +/- 1.1 X 10(-10) M, n = 8). Treatment of castrated males with dihydrotestosterone (DHT) for 24 h (250 micrograms/100 g of body weight) increased the Kd of HPA cytosol only slightly (1.6 X 10(-10) M, mean of two replicates). Scatchard analysis of salt-extracted nuclear androgen receptor (ARn) showed a single, high-affinity binding site with similar Kd values in PIT and HPA of intact and castrated, DHT-treated male rats (PIT Kd = 7.3 X 10(-10) M, 9.3 X 10(-10) M; HPA Kd = 1.5 X 10(-9) M, 1.3 X 10(-9) M, respectively). Competition studies involving a range of several radioinert steroids revealed that the binding of [3H]-R1881 to cytosol (ARc) and nuclear extract was specific for androgen receptor when triamcinolone acetonide (10 microM) was added. The ARc and ARn levels were quantified in PIT, preoptic area (POA), hypothalamus (HT), amygdala, hippocampus, and cortex by single point estimation. Significantly (p less than 0.01) greater amounts of ARc were detected in PIT of ovariectomized females (32.7 +/- 2.9 fmol/mg of protein) than in that of orchidectomized males (22.33 +/- 1.6 fmol/mg of protein). The highest levels in the brain were seen in HT and POA. Pituitary ARc in females varied throughout the estrous cycle. Significantly (p less than 0.01) greater amounts were detected on estrus (45.8 +/- 2.2 fmol/mg of protein) and proestrus (39.0 +/- 1.9 fmol/mg of protein) than on diestrus (29.2 +/- 1.5 fmol/mg of protein). These data confirm the existence of specific receptors for androgen in male and female brain and PIT, and suggest an important role for androgen in the control of PIT hormone secretion in the female.     

Effects of lysine-vasopressin (LVP) and 1-deamino-8-D-arginine-vasopressin (dDAVP) upon electrical potential, short-circuit current and transepithelial D.C. resistance of the frog skin

The synthetic analogue of vasopressin, 1-deamino-8-D-arginine-vasopressin (dDAVP), possesses a protracted antidiuretic activity while having practically no pressoric activity as compared to arginine-vasopressin (AVP) or lysine-vasopressin (LVP). The effects of LVP and dDAVP were studied on the frog skin (Rana temporaria) sodium transport as reflected by the short-circuit current (SCC) level, on an Ussing apparatus. The application two different equimolar doses of LVP or dDAVP (approx. 9.4 X 10(-8) mol X l-1 and 18.8 X 10(-8) mol X l-1 to the inner surface of the skin resulted in identical maximal increases of sodium transport. However, the maximum transport stimulation after the application of dDAVP was delayed by about 30 min as compared to the stimulation by LVP (P less than 0.01). In addition, a protracted recovery of SCC towards its original levels was observed in experiments with dDAVP application after the hormone removal (P less than 0.01). It is concluded that dDAVP stimulates Na+ transport through the frog skin despite its lacking pressoric activity. Thus, the natriferic activity of vasopressin is related to its antidiuretic rather than pressoric activity. Maximum increase in the sodium transport following dDAVP application was delayed and more protracted as compared to the effect of LVP.     

Sodium transport and oxygen consumption in toad bladder. A thermodynamic approach.

The relationship between active sodium transport and oxygen consumption was investigated in toad urinary bladder exposed to identical sodium-Ringer's solution at each surface, while controlling the transepithelial electrical potential difference delta phi. Rates of sodium transport and oxygen consumption were measured simultaneously, both in the short-circuited state (delta phi = 0) and when delta phi was varied. Under short-circuit conditions, when the rates of active sodium transport changed spontaneously or were depressed with amiloride, the ratio of active sodium transport to the estimated suprabasal oxygen consumption Na/O2 was constant for each tissue, but varied among different tissues. Only when delta phi was varied did the ratio Na+/O2 change with the rate of active sodium transport; under these circumstances dNa+/dO2 was constant but exceeded the ratio measured at short-circuit [(Na+/O2)delta phi = 0[. This suggests that coupling between transport and metabolism is incomplete. The results are analyzed according to the principles of nonequilibrium thermodynamics, and intepreted in terms of a simple model of the transepithelial sodium transport system.     

Discrimination between subclasses of human high-density lipoproteins by the HDL binding sites of bovine liver

The binding of human 125I-labeled HDL3 (high-density lipoproteins, rho 1.125-1.210 g/cm3) to a crude membrane fraction prepared from bovine liver closely fit the paradigm expected of a ligand binding to a single class of identical and independent sites, as demonstrated by computer-assisted binding analysis. The dissociation constant (Kd), at both 37 and 4 degrees C, was 2.9 micrograms protein/ml (approx. 2.9 X 10(-8) M); the capacity of the binding sites was 490 ng HDL3 (approx. 4.9 pmol) per mg membrane protein at 37 degrees C and 115 at 4 degrees C. Human low-density lipoproteins (LDL) and very-low-density lipoproteins (VLDL) also bound to these sites (Kd = 41 micrograms protein/ml, approx. 6.7 X 10(-8) M for LDL, and Kd = 5.7 micrograms protein/ml, approx. 7.0 X 10(-9) M for VLDL), but this observation must be considered in light of the fact that the normal circulating concentrations of these lipoproteins are much lower than those of HDL. The binding of 125I-labeled HDL3 to these sites was inhibited only slightly by 1 M NaCl, suggesting the presence of primarily hydrophobic interactions at the recognition site. The binding was not dependent on divalent cations and was not displaceable by heparin; the binding sites were sensitive to both trypsin and pronase. Of exceptional note was the finding that various subclasses of human HDL (including subclasses of immunoaffinity-isolated HDL) displaced 125I-labeled HDL3 from the hepatic HDL binding sites with different apparent affinities, indicating that these sites are capable of recognizing highly specific structural features of ligands. In particular, apolipoprotein A-I-containing lipoproteins with prebeta electrophoretic mobility bound to these sites with a strikingly lower affinity (Kd = 130 micrograms protein/ml) than did the other subclasses of HDL.     

Binding and second messengers of prostaglandins F2 alpha and E1 in primary cultures of rabbit endometrial cells

Several factors and hormones are thought to play a role in the growth control of endometrial cells. We have shown that prostaglandin F2 alpha (PGF2 alpha) is a growth factor for primary cultures of rabbit endometrial cells grown in serum-free, chemically defined medium and that prostaglandin E1 (PGE1) antagonizes the PGF2 alpha induction of growth (Orlicky et al., 1986). [3H]PGF2 alpha binds to whole cells in a time (optimal approximately 30 min)- and temperature-dependent (optimal 37 degrees C), disassociable (90% disassociable within 30 min), saturable (Kd1 = 4.9 X 10(-8) M, n1 = 1.2 X 10(5) molecules/cell; Kd2 = 2.6 X 10(-7) M, n2 = 3.0 X 10(5) molecules/cell), and specific manner. [3H]PGE1 binds in a time-dependent (optimal 25 min), disassociable (90% disassociable within 10 min), saturable (Kd = 6.4 X 10(-8) M, n = 1.2 X 10(5) molecules/cell), and specific manner. This specific binding of [3H]PGF2 alpha and [3H]PGE1 is down-regulatable by prior treatment of the cultures with unlabeled ligand, and up-regulatable by prior treatment of the cultures with indomethacin to inhibit endogenous PG synthesis. Proteolytic enzyme treatment for 2 min reduces the specific binding of PGF2 alpha by 75%. PGE1 stimulates intracellular cAMP synthesis and accumulation in a time (optimal 10 min)- and concentration (half-maximal stimulation at 10(-6) M)-dependent manner but has no effect on intracellular cGMP. PGF2 alpha has no effect on either intracellular cAMP or cGMP in this system. We describe here for the first time the analysis at a biochemical level of the interaction between two prostaglandins, antagonistic to each other in terms of growth regulation.