Active sodium and potassium transport in high potassium and low potassium sheep red cells.

The kinetic characteristics of the ouabain-sensitive (Na + K) transport system (pump) of high potassium (HK) and low potassium (LK) sheep red cells have been investigated. In sodium medium, the curve relating pump rate to external K is sigmoid with half maximal stimulation (K(1/2)) occurring at 3 mM for both cell types, the maximum pump rate in HK cells being about four times that in LK cells. In sodium-free media, both HK and LK pumps are adequately described by the Michaelis-Menten equation, but the K(1/2) for HK cells is 0.6 +/- 0.1 mM K, while that for LK is 0.2 +/- 0.05 mM K. When the internal Na and K content of the cells was varied by the PCMBS method, it was found that the pump rate of HK cells showed a gradual increase from zero at very low internal Na to a maximum when internal K was reduced to nearly zero (100% Na). In LK cells, on the other hand, no pump activity was detected if Na constituted less than 70% of the total (Na + K) in the cell. Increasing Na from 70 to nearly 100% of the internal cation composition, however, resulted in an exponential increase in pump rate in these cells to about (1/6) the maximum rate observed in HK cells. While changes in internal composition altered the pump rate at saturating concentrations of external K, it had no effect on the apparent affinity of the pumps for external K. These results lead us to conclude that the individual pump sites in the HK and LK sheep red cell membranes must be different. Moreover, we believe that these data contribute significantly to defining the types of mechanism which can account for the kinetic characteristics of (Na + K) transport in sheep red cells and perhaps in other systems.

Suppression of pituitary-gonadal function by a potent new luteinizing hormone-releasing hormone antagonist in normal men.

LHRH antagonists compete with endogenous LHRH for binding to receptors on pituitary gonadotrophs and thereby inhibit gonadal function by suppressing gonadotropin secretion. We studied the effects of a recently developed LHRH antagonist on the pituitary-gonadal axis in man. The antagonist Detirelix [( N-Ac-D-Nal(2)1, D-pCl-Phe2,D-Trp3, D-hArg(Et2)6, D-Ala10]LHRH) was given as a single sc injection to nine normal men at three dose levels (5, 10, and 20 mg) at intervals of at least 7 days. Serum FSH, LH, and testosterone levels were measured before treatment, at frequent intervals for 48 h, and 72, 96, and 168 h after administration of the antagonist. Mean serum FSH levels decreased (P less than 0.001) from 6.9 +/- 0.5 (+/- SEM) mIU/mL to nadirs of 4.4 +/- 1.1, 3.6 +/- 0.9, and 4.1 +/- 0.9 after the 5-, 10-, and 20-mg doses, respectively. Serum LH levels decreased (P less than 0.001) from 6.2 +/- 0.3 mIU/mL to nadirs of 3.3 +/- 0.4, 2.8 +/- 0.3, and 2.7 +/- 0.3 after all three doses. Serum testosterone levels decreased (P less than 0.001) in a dose-dependent fashion from 5.1 +/- 0.2 ng/mL to nadirs of 1.3 +/- 0.3, 0.9 +/- 0.3, and 0.6 +/- 0.1 after the same doses. After the initial testosterone decrease, however, escape occurred 12-28 h after the lower doses. The area under the response curve, describing hormone concentrations as a function of time during the study, diminished by 23 +/- 2%, 36 +/- 4%, and 36 +/- 3% for FSH, by 14 +/- 6%, 30% +/- 6%, and 34 +/- 5% for LH, and by 41 +/- 5%, 58 +/- 6%, and 68 +/- 4% for testosterone with the same doses, respectively. The apparent plasma disappearance half-life of Detirelix by RIA was at least 41 h after all three doses. Detirelix elicited only a minor local reaction; no systemic side-effects were observed within the dose range used. These results indicate that this LHRH antagonist is a safe, highly potent inhibitor of the human pituitary-gonadal axis with an exceptionally long duration of action.

Hepatomas: hormone receptors and therapy.

Prompted by studies that suggested a causal relationship between sex steroids and human liver cancer, we assayed specimens of hepatoma from five patients and adjacent liver tissue from three of the same patients for estrogen receptors. After finding that the assay material contained specific cytosolic receptor proteins for estradiol, we treated a second group of five patients who had hepatomas with progestin. This therapy resulted in tumor regression in two of these patients.

Sex steroid receptors in gynecologic cancer.

Steroid hormone receptors provide the means by which estrogen and progesterone influence their target tissues. Receptor assays have proved clinically useful in the management of breast cancer, and current research indicates they may be valuable in managing some gynecologic neoplasms. This is a review of the current and potential importance of receptors in gynecologic oncology.

Hormone receptors in renal cell carcinoma. Their utility as predictors of response to endocrine therapy.

We have performed 23 estrogen and 24 progesterone receptor assays on tumor specimens from 26 patients with renal cancer, ten of whom subsequently received hormonal therapy for metastatic disease. Only one of these specimens contained measurable estrogen receptor levels, three contained low, but measurable progesterone receptor levels, and the remaining specimens contained no measurable estrogen or progesterone receptors. None of ten patients with metastases showed evidence of an objective response to hormonal therapy. Our data suggest that few, if any, renal cancers have high titers of estrogen or progesterone receptors, and that those patients whose tumors have low receptor titers are unlikely to respond to hormonal therapy.

Recurrent macromastia after subcutaneous mastectomy.

Management of sudden unrelenting breast growth in a young woman included use of antiestrogen hormone therapy and subcutaneous mastectomy. Later, massive breast growth again occurred during pregnancy, requiring a repeat postpartum subcutaneous mastectomy. The dramatic response to a specific antiestrogen agent and the subsequent massive regrowth of breast tissue after subcutaneous mastectomy suggests that breast tissue is extremely sensitive to circulating hormones in certain patients with macromastia. The unusual nature of this patient's recurrent macromastia warrants this review of reports of similarly affected patients and discussion of general concepts in the medical and surgical management of the disorder.

Estrogen and thyroid-stimulating hormone (TSH) receptors in neoplastic and nonneoplastic human thyroid tissue.

Estrogen-binding receptors (ER) and thyroid-stimulating hormone (TSH) receptors were observed in the cytosol and in a membrane particulate fraction, respectively, in most neoplastic and nonneoplastic human thyroid tissues. Fourteen of 15 thyroid neoplasms and 6 of 15 nonneoplastic thyroid specimens had estrogen receptors (assuming the sensitivity of our estrogen receptor assay is 0.2 fmole/mg protein), and 14 of 15 thyroid neoplasms and 11 of 15 nonneoplastic thyroid specimens had a high affinity, low capacity TSH receptor. Neoplastic thyroid tissue had more ER (2.35 +/- 0.70/fmole/mg protein) than nonneoplastic thyroid tissue (0.57 +/- 0.181/fmole/mg protein) removed from the same patients (P less than 0.05). The Kd for ER did not differ in nonneoplastic (0.41 +/- 0.090 nM) and neoplastic (0.311 +/- 0.048 nM) thyroid tissue. The number of TSH receptors was comparable in neoplastic (0.609 +/- 0.191 pmole/mg protein) and in nonneoplastic (0.765 +/- 0.181 pmole/mg protein) thyroid tissue removed from the same patients who had the ER studies. The maximal adenylate cyclase response to TSH was greater in the neoplastic (147 +/- 26.9 pmole/mg protein/30 min) than in nonneoplastic thyroid tissue (32.8 +/- 6.69 pmole/mg protein/30 min) (P less than 0.001) suggesting a greater metabolic responsiveness of the neoplastic thyroid tissue to TSH. No correlation was evident, however, between the number of estrogen and TSH receptors in nonneoplastic and neoplastic thyroid tissue (r = 0.226). This study demonstrates that neoplastic human thyroid tissues have both estrogen receptors and TSH receptors. The neoplastic tissue also has a greater AC response to TSH than nonneoplastic thyroid tissue.

Hormone receptors in hepatoblastoma: a demonstration of both estrogen and progesterone receptors.

A case of mixed hepatoblastoma occurring in a four-year-old girl is described. Light and electron micrographs illustrate a predominant mesenchymal component and a less conspicuous epithelial element. The tumor contained specific cytosolic protein receptors for estrogen and progesterone. The estrogen receptor titer was 10.5 fmol/mg cytosol protein and the progesterone receptor titer was 62.2 fmol/mg cytosol protein.

Progesterone receptors: saturation analysis by a solid phase hydroxylapatite adsorption technique.

An assay for cytoplasmic progesterone receptor is described in which receptor is bound to hydroxylapatite and is washed before saturation analysis. Using 3H-progesterone as ligand, the assay yields lower, more reproducible nonspecific binding than is obtained with dextran-coated charcoal techniques and may provide a clinically useful method of measuring progesterone receptor in breast carcinoma. Nuclear progesterone receptors can also be determined by the same technique.

Regulation of secretion from the adrenal medulla. Evidence for adenylate cyclase activity in secretory vesicle membranes.

Adenylate cyclase activity has been found in purified secretory vesicle membranes from the adrenal medulla. Activity was detected both by formation of radioactive cAMP from [alpha-32P]ATP and by the competitive protein binding assay for cAMP. Activity was highest at pH 8.0 to 8.5, and was stimulated by sodium fluoride and GppNHp, a GTP analogue known to stimulate adenylate cyclase activity in plasma membrane preparations. The reaction rate was strongly dependent on the molar ratio of Mg2+:ATP in the system. This is the first demonstration of adenylate cyclase in a secretory vesicle membrane.

Regulation of the transmembrane potential of isolated chromaffin granules by ATP, ATP analogs, and external pH.

The transmembrane potential of isolated chromaffin granules has been measured using the permeant ions [14C]methylamine and [35S]thiocyanate, as well as the fluorescent probe, 9-aminoacridine. At pH 7.0, the granule membrane had a Nernst proton potential of -45mV, inside negative. This potential was sensitive to the external pH, but was unaffected by K+,Na+, Ca2+, Mg2+, or other cations. The pH of zero potential was 6.25 for both methylamine and thiocyanate. Thiocyanate also had a Nernst potential of similar magnitude and sign to that of methylamine at pH 7.0, and was also sensitive to variation in external pH. Mg2+ATP was found to depolarize the granule membrane by a saturable mechanism with a K 1/2 for ATP of 40 muM. Ca2+ was only 30% as effective as Mg2+ in supporting the ATP effect. The pH optimum for this process was 6.25 and appeared to be accompanied by a marked alkalinization of the granule interior. The specificity for ATP was further tested with structural analogs of ATP and GTP. The rate of change of membrane potential in response to changes in external pH or Mg2+ATP was estimated using the fluorescent probe 9-aminoacridine. Changes came to completion in less than 1 s. This suggested that the ATP effects were not dependent on an enzymatic transformation but on an ATP-induced conformational change in the membrane. We conclude that the chromaffin granule exists in at least two proton permeability states, corresponding to the presence or absence of Mg2+ATP. These states may be related to hormone release from granules and regulation of secretion in vivo.

ATP-stimulated transmitter release and cyclic AMP synthesis in isolated chromaffin granules.

ATP stimulates chromaffin granules from the bovine adrenal medulla to release epinephrine and specific soluble proteins. ATP analogs substituted in the beta-gamma position with either nitrogen or carbon were also found to be effective at inducing release from isolated chromaffin granules. However, an ATP analog substituted at the alpha-beta position with carbon was strongly inhibitory. Cyclic AMP was also found to be synthesized by isolated chromaffin granules under release conditions. ATP analogs were effective as substrates for adenylate cyclase in the same order as their efficiency for inducing release from vesicles. Hydrolysis at the beta-gamma linkage of ATP therefore is probably not necessary for release; however, hydrolysis at the alpha-beta position may be important in the release process. Cyclic AMP may be produced and play a regulatory role in this event.

Regulation of release from isolated adrenergic secretory vesicles by ATP-mediated changes in transmembrane potential and anion permeability.

Isolated chromaffin granules release their contents when exposed to calcium, magnesium, ATP, and high levels of chloride ions. The mechanism of release is not well-understood, but changes in anion permeability may be involved. We found that another anion, thiocyanate (SCN-), also activated release in a fashion similar to chloride, while isethionate (HO-CH2-CH2SO3-) an impermeant anion, was inactive. Mg++-ATP was found to activate the uptake of 36Cl and 14C-SCN, leading us to conclude that activation of anion uptake might be involved in the release process. The 36Cl and the 14C-SCN compartments were then compared by studying displacement of the trace anions by excess cold mass. Chloride and SCN displaced large amounts of both 36Cl and 14C-SCN, while isethionate displaced little of either tracer anion. We suggest, on the basis of these data, that ATP-mediated anion uptake may be the basis for the release mechanism. Release may occur as a consequence of anion and subsequent water uptake into granules, resulting in osmotic imbalance and osmotic shock. This may also be of physiologic importance, and we propose a cellular model for secretion based on the biochemical properties of the isolated chromaffin granule.

Stimulation of active potassium transport in LK sheep red cells by blood group-L-antiserum.

Anti-L serum prepared by immunization of a high-potassium-type (HK) (blood type MM) sheep with blood from a low-potassium-type (LK) (blood type ML) sheep contained an antibody which stimulated four- to sixfold K(+)-pump influx in LK (LL) sheep red cells. In long-termin vitro incubation experiments, LK sheep red cells sensitized with anti-L showed a net increase in K(+) after two days of incubation at 37°C, whereas HK-nonimmune (NI)-serum-treated control cells lost K(+). The antibody could be absorbed by LK (LL) sheep red cells but not by HK sheep red cells. Kinetic experiments showed that the concentration of external K(+) ([K(+)]0) required to produce halfmaximum stimulation of the pump ([Na(+)]0=0, replaced by Mg(++)) was the same (0.25 mM) in L-antiserum-treated or untreated LK cells. LK cells with different [K(+)]i (Na(+) replacement) were prepared by the p-chloromercuribenzene sulfonate (PCMBS) method. At [K(+)]0=5 mM, pump influx decreased as [K(+)]i increased from 1 to 70 mM in L-antiserum-treated LK cells, whereas LK cells treated with HK-NI-serum ceased to pump at [K(+)]i=35 mM. Exposure to anti-L serum produced an almost twofold increase in the number of pump sites of LK cells as measured by the binding of tritiated ouabain by LK sheep red cells. These findings indicate that the formation of a complex between the L-antigen and its antibody stimulates active transport in LK sheep red cells both by changing the kinetics of the pump and by increasing the number of pump sites.

Estrogen receptors in male breast cancer: clinical and pathologic correlations.

Breast cancer tissue from 12 male patients was evaluated for the presence of estrogen receptors (ER), and at least 10 of 12 were found to be high in ER (ER+). A combination of previously published cases and our own experience indicates that 80% of patients (37/47) have ER+ tumors. This incidence of ER positivity in men is greater than that in women with breast cancer. Clinical responses to hormonal therapy have been observed in the ER+ patients but not in one ER--patient. Measurement of ER may be clinically important in men with breast cancer.

Combined modality therapy for Stage IIIMO non-small cell lung cancer. A five-year experience.

Between 1975 and 1980, 101 patients with inoperable Stage IIIMO non-small cell lung carcinoma were entered into combined radiotherapy and chemotherapy trials at Michael Reese Hospital and University of Chicago Hospital. Sixty-four percent of the patients responded. Median survival for all patients was 8.8 months. Responders survived 13.7 months and nonresponders 4.6 months (P = 0.002). Patients treated with 4200 rad had a higher response rate than those treated with 3000 rad (74% versus 54%, P = 0.04) but there was no difference in survival. Although all patients with squamous cell carcinoma died by 30 months, 18% of patients with adenocarcinoma and 20% of patients with large cell carcinoma are long-term survivors. Brain metastases occurred more frequently in patients with large cell or adenocarcinoma than in patients with squamous cell carcinoma (P = 0.02). The prognostic effect of age, initial performance status, sex, histology, and tumor extent are examined. Toxicity was substantial with a 13% treatment-related mortality. Combined modality therapy may benefit selected patients with non-squamous cell types, but more effective chemotherapeutic agents are needed. Prophylactic cranial irradiation in patients with large cell carcinoma or adenocarcinoma may decrease the incidence of subsequent brain metastases.