Regulatory elements in the human immunodeficiency virus type 1 long terminal repeat LTR (HIV-1) responsive to steroid hormone stimulation.

Within the long terminal repeat (LTR) of the human immunodeficiency virus type 1 (HIV-1) provirus there exists a steroid hormone-responsive element corresponding to the TGTTCT sequence identified as the glucocorticoid receptor binding element within the LTR of mouse mammary tumor virus. We have used an LTR(HIV-1)-chloramphenicol acetyl transferase (CAT) plasmid construct to transfect infected H9V3 and noninfected H9 cells. Four hours before harvest the cells were divided into two parts and half was treated with hydrocortisone (10(-7) M). The cells were harvested and washed, and the CAT activity was measured. In eight repeat experiments an increased expression of the CAT gene has consistently been observed in H9V3 cells in response to the glucocorticoid but no significant effect of the steroid was observed in noninfected cells. Double transfection of LTR(HIV-1)-TAT and LTR(HIV-1)-CAT into noninfected H9 cells results in a cell population in which the CAT gene was responsive to glucocorticoid stimulation. A time course and dose response for the steroid effect have been determined and the binding of steroid receptor fo the LTR-DNA characterized by gel retardation experiments.

Characteristics of the calf uterine androgen receptor.

The highest molecular weight form of the calf uterine androgen receptor separates as an 11S form in glycerol gradients. This "cytosolic" receptor, prepared in the presence of molybdate, polyethyleneimide and low ionic strength, dissociates into 9S and 7.2S forms with increasing KCl concentration. A 4.5S androgen binding component appears as the predominant form of the receptor in the absence of polyethyleneimide and this unit quantitatively converts to a stable 3.5S form in the absence of molybdate. Renaturation of partially purified protein, separated by SDS-PAGE electrophoresis, demonstrates the presence of an androgen binding component in the 110 kDa region of the gel. This renatured protein separates as a 4.5S component in glycerol gradients and has a Stokes radius of 6 nm. Photoaffinity labelling of partially purified receptor preparations, followed by SDS-PAGE electrophoresis, reveals the presence of an androgen binding component having a molecular weight of 115 kDa. The binding characteristics and specificity of the receptor binding to R1881 have been studied and a DHT-affinity chromatography resin used to purify the receptor.

Role of cyclic AMP in steroid action in rat intestine.

We have investigated the effect of mineralocorticoids on intestinal fluid and electrogenic glucose-linked Na+ transport across isolated sections of the rat small intestine. A rapid in vitro response is observed that contrast with the delay normally associated with steroid hormone responses. Cyclic AMP is known to affect intestinal glucose, water and Na+ transport and the effects of the steroids may be understood in terms of an inhibitory effect on intestinal cyclic AMP production. An inhibitory effect of the steroids on membrane-bound adenylate cyclase has been demonstrated and dose-response effects suggest the presence of specific membrane-bound glucocorticoid receptors.

Developmental aspects of steroid-induced ammonia release from isolated sections of rat intestine.

Release of ammonia from isolated intestinal sections of adult male rats is higher than that measured using immature animals. The increase appears to be Na+ dependent and develops during the spurt of growth at puberty. Developmental changes in Na+-dependent ammonia release from isolated sections of the intestine and growth of the small intestine in male and female rats have been compared. Intestinal growth increases far more rapidly than body weight and in the males critical developmental changes occur early during weaning and during puberty. In females the major change is at weaning and little further change occurs during puberty. Treatment of young animals with aldosterone or testosterone increases the Na+-dependent ammonia release precociously. Dose-response effects of testosterone and aldosterone in distal sections of the small intestine have been compared.

Effects of saline exposure on the response of toad bladder (Bufo marinus) to aldosterone.

1. The short-circuit current response of toad bladders to high concentrations of aldosterone (2 x 10(-7) M) has been investigated following saline exposure of toads. 2. Limited saline exposure appears to enhance the aldosterone-stimulated response. 3. Additional saline exposure results in a reduced aldosterone response. 4. Overnight pre-incubation of isolated bladders proved a better pretreatment condition for study of an aldosterone short-circuit current response. 5. Plasma aldosterone concentrations and studies of [3H]aldosterone binding in the bladders have failed to demonstrate significant effects of saline exposure on endogenous aldosterone concentrations.

Aldosterone effects on renal metabolism.

1. Manometric studies of the sodium dependent oxygen consumption in rat kidney slices have failed to reveal any significant effect of aldosterone treatment, adrenalectomy or sodium diet on sodium diet on sodium metabolism. 2. However, ammonia release from kidney slices was significantly reduced following adrenalectomy and this decrease was influenced by aldosterone treatment. 3. The dose-response characteristic obtained for this aldosterone-stimulated ammonia release has been determined. 4. The effect of a high Na+ diet on the ammonia release has been studied. An initial decrease after 2 days may be associated with decreased endogenous aldosterone secretion. However, aldosterone (2.5 microgram/100 g body weight)injections into these high Na+ treated animals fails to restore the normal ammonia release. 5. The effects of aldosterone (2.5 microgram/100 g body weight), dexamethasone (2.5 microgram/100 g body weight) and corticosterone (2.5 microgram/100 g body weight) injections, in adrenalectomized rats, on ammonia release and tissue tyrosine aminotransferase activities have been compared.

Role of mitochondrial Ca2+ in antidiuretic hormone action.

The calcium ion concentration measured in rat kidney mitochondria, isolated from vasopressin treated tissue, has a dose response characteristic in which the calcium concentration reached a minimum at low doses of vasopressin (2 mU/ml), at higher doses of hormone the mitochondrial calcium ion concentration increases reaching a value close to that of the controls with vasopressin (100 mU/ml). This efflux and subsequent uptake of mitochondrial calcium has been shown to be a direct effect of the varying cyclic AMP concentrations. Sodium and water permeability effects of vasopressin have been shown in toad bladder to have different dose response characteristics. Maximum sodium transport occurs at a lower dose of vasopressin (2 mU/ml) and is believed to be associated with direct permeability effects of the hormone. Maximum water transport occurs at a higher dose of vasopressin (100 mU/ml) over a concentration range associated with hormone-stimulated adenylate cyclase activity. The water transport response to low doses of vasopressin may be potentiated by aldosterone treatment, an effect that can be related to the inhibition of tissue phosphodiesterase activity and subsequent increased cyclic AMP concentrations. In steroid depleted conditions the cyclic AMP medicate efflux of mitochondrial calcium ions, that occurs at low doses of vasopressin, may prevent the release of membrane bound calcium ions and thus inhibit the water permeability effect of the hormone. Higher levels of cyclic AMP reverse this inhibitory effect and give rise to an increased water flow. It is concluded that cyclic AMP and intracellular concentrations of calcium ion act as inter-related mediators of antidiuretic hormone action.

Effect of steroid depletion on the response of toad bladder to vasopressin.

1. We have investigated the water transport and short-circuit current (s.c.c.) response to vasopressin (1 mu./ml. and 100 mu./ml.) in isolated toad urinary bladders (Bufo marinus) following overnight incubation in the presence or absence of steroid-containing Ringer solution. 2. The water transport response to the lower dose of vasopressin (1 mu./ml.) was considerably reduced in 'steroid depleted' conditions, wheras the response to the higher dose of vasopressin (100 mu./ml.) was not similarly affected. 3. Aldosterone 3. Aldosterone 3. Aldosterone (10(-7)M) potentiated the water transport response to the lower dose of vasopressin (1 mu./ml.) but had no effect on the response to the higher dose (100 mu./ml.). 4. There was no effect of 'steroid depletion' or aldosterone treatment on the vasopressin s.c.c. response when measured as a percentage increase above basal levels. 5. In 'steroid depleted' conditions vasopressin (1 mu./ml.) maximally stimulated Na+ transport (s.c.c.) but a higher dose of vasopressin (100 mu./ml.) was required for maximum water transport. 6. We have failed to obtain any potentiation effect of corticosterone (10(-7)M) on the water transport or s.c.c. response to vasopressin (1 mu./ml.).