An approach to the identification of adenosine''s inhibitory site on adenylate cyclase

The separate and combined effects of molybdate and dithiothreitol on the stability of human uterine 9 S estrogen receptor were studied. Maximal, short-term, protection of the 9 S estrogen receptor was achieved by the joint inclusion of both stabilizing agents in cytosol buffers. This molybdate—dithiothreitol-mediated stability was dependent on reducing agent concentration inferring sulphydryl involvement in 9 S receptor protection by molybdate. The study also showed that molybdate—dithiothreitol could not prevent the gradual decay of the 9 S estrogen receptor to the 4 S form in cytosols stored at 4°C over prolonged periods.     

Pregnancy termination by prostaglandin F2α stimulates maternal behavior in the rat

Treatment with PGF_2α resulted in the termination of pregnancy in 16- and 19-day pregnant rats, but not in 10- or 13-day pregnant rats. Rats that aborted displayed a rapid onset of maternal behavior when tested with foster pups. Aborted rats also displayed sexual receptivity and ovulation: these phenomena resemble the sequence of events following hysterectomy on the same days of pregnancy. Both can be related to the events surrounding normal parturition in the rat. The results are interpreted as due to a pregnancy-induced deactivation of the factor in the uterus that prevents estrogen from stimulating maternal behavior in nonpregnant females. In the absence of this factor, the PGF_2α-induced rise in estrogen secretion facilitates maternal behavior and sexual behavior and induces ovulation.     

Kinetic studies of the applicability of the common site concept to the 3β-hydroxysteroid dehydrogenase: 5-ene-3-ketosteroid isomerase activities of human placental microsomes

Pregnenolone (3β-hydroxy-5-pregnen-20-one) and DHA (3β-hydroxy-5-androsten-17-one), substrates for 3β-hy-droxysteroid dehydrogenase (3β-HSD), with K_M values of 15–40 nM, were ineffective inhibitors of 5-ene-3-ketosteroid isomerase (isomerase), with K_I values >40 μM in each case. Progesterone and androstenedione (4-androstene-3, 17-dione), 3β-HSD inhibitors with K_I values of 5.0 μM and 0.8 μM respectively, were also relatively ineffective inhibitors of isomerase, with K_I values of 30 μM and 16.5 μM respectively. Exposure of microsomes to hydrogen peroxide, which significantly increases the K_M for pregnenolone as a 3β-HSD substrate, had no effect on the K_M for the isomerase substrate 5-pregnene-3, 20-dione.It is concluded that the data do not support the common site concept with regard to the conversion of pregnenolone to progesterone by human placental microsomes.     

Thyroid hormone modulation of agonist - beta-adrenergic receptor interactions in the rat heart

We have investigated alterations in beta-adrenergic receptors in rat myocardial membranes derived from hypothyroid and hyperthyroid animals. (-)Isoproterenol competition curves with (-)[³H]dihydroalprenolol revealed that isoproterenol binds to the beta-adrenergic receptor with two distinct affinity states having high (K_H) and low (K_L) dissociation constants. In the presence of guanine nucleotides the isoproterenol competition curve steepened and had a higher EC₅₀ (50% displacement). This was due to a transition of the high affinity state to a uniformly low affinity state. Using computer modeling of these competition curves, we have demonstrated that in hyperthyroidism, the isoproterenol curve in the absence of guanine nucleotides is shifted to the left with the EC₅₀ changing from 180 ± 40 to 80 ± 20 nM (p < .02). The fold shift (4 fold) in K_H (nM) 30 ± 9 to 7 ± 2 (p < .001) is greater than that (1.6 fold) in K_L (nM) 595 ± 56 to 376 ± 34 (p < .001) such that the K_L/K_H ratio shifted from 20 ± 3 to 54 ± 9 (p < .001). The ratio, K_L/K_H, for a particular agonist appears to be related to its efficacy in activating adenylate cyclase.There was no significant alteration in any of these parameters in hypothyroid animals. Receptor number was decreased in hypothyroidism, 16 ± 3 fmol/mg protein (p < .03) and increased in hyperthyroidism 44 ± 4 (p < .03) compared to control 26 ± 2.In the rat heart agonist affinity and receptor number are modulated in hyperthyroidism, but only receptor number in hypothryoidism. Thus thyroid hormone can modify not only receptor number but agonist affinity as well.     

A DNA-binding fraction of mouse kidney 3-ketosteroid reductase: Comparison with androgen receptors

Since approximately 1% of 3-ketosteroid reductase (which metabolizes dihydrotestosterone [17β-hydroxy-5α-androstan-3-one] to 5α-androstane-3α,17β-diol or 5α-androstane-3α,17β-diol) from mouse kidney cytosol adheres to DNA under conditions that allow virtually complete androgen receptor binding, these two DNA-binding activities were compared in cytosol extracts of mouse kidney and hypothalamus-preoptic area. This DNA-binding fraction of 3-ketosteroid reductase was distinguished from androgen receptor in several ways: (1) its pattern of elution from DNA-cellulose with steps of increasing NaC1 concentration differed from that for receptors from wild-type kidney; (2) it was influenced differently by the mutation Tfm, both in level and in DNA-cellulose elution pattern; (3) in mouse kidney cytosol it was relatively stable at moderate (25°C) temperatures which rapidly inactivated ligand-free androgen receptors in the same cytosols; (4) the DNA-binding was not proportional to androgen receptor levels between two wild-type tissues, the hypothalamus-preoptic area and kidney. By these criteria, a simple relationship of androgen receptors and a DNA-binding fraction of 3-ketosteroid reductase activity is unlikely.     

Primary and secondary autologous mixed-lymphocyte interactions in mice occur via antigens encoded by genes in the I region of the major histocompatibility complex

The antigens stimulating the autologous mixed-lymphocyte reactions (AMLR) were studied in experiments employing specific blocking antisera and through panels of secondary stimulating cells differing in the MHC. Antisera directed to the entire I region, I-A, I-EC, or A?Eα were all effective in blocking the primary AMLR. A principal role for those determinants encoded in I-A was indicated by secondary stimulation experiments. In secondary cultures, I-EC encoded determinants generated weaker responses in the absence of I-A homology.     

BCG-induced granulomatous pulmonary inflammation and splenomegaly in mice: A T-cell-dependent response

When C57BL/6 mice were injected iv with BCG in an oil-in-saline emulsion, they developed intense pulmonary granulomatous inflammation (PGI) and splenomegaly as well as chemotactic activity for macrophages and angiotensin-converting enzyme (ACE) in their lung fluids. PGI, splenomegaly, and levels of chemotactic activity and ACE were markedly reduced in T-cell-deficient “B” mice. The capacity to develop PGI was fully restored and splenomegaly was partially restored in “B” mice by the provision of syngeneic thymocytes, spleen cells, or purified T cells. These results indicate that the full expression of BCG-induced PGI is dependent upon thymus-derived cells and is associated with high levels of chemotactic activity for macrophages and ACE in the lung lavage fluid. Although BCG-induced splenomegaly appears to be T cell dependent, it did not reach its full magnitude in reconstituted “B” mice.