Vinod K. Bhalla

Gonadal receptors. I. Evidence for irreversibility in the binding of human chorionic gonadotropin and human luteinizing hormone.

The binding of human chorionic gonadotropin and human luteinizing hormone to particulate receptors of rat testes has generally been assumed to follow an equilibrium model similar to that proposed for many enzyme systems. Our work shows that equilibrium dissociation constant (Kd) and number of hormone binding sites (Bmax) are highly sensitive to changes in hormone and/or receptor concentration and to treatment received by tissue or receptor preparation prior to the assay. The results of binding assays obtained using receptor preparation pretreated with hormone (labeled as well as unlabeled) indicated that the binding reaction between hormone and receptor was irreversible and that pretreatment of the tissue with hormone greatly alters the number of high affinity gonadotropin binding sites in the testicular homogenate. Data from studies involving increasing receptor concentrations revealed that increasing the mass of particulate receptors in the binding assays leads to higher Kd as well as Bmax values. These findings are incompatible with a binding model based upon occupancy of receptor sites and the state of equilibrium implied. The incompatibilities are analyzed and an alternate model advanced (Bhalla, V.K., Trowbridge, C.G., Chen, C.J.H., Lindeman, J.G. and Rojas, F.J. (1979) Biochim. Biophys. Acta 584, 436--453).

Effects of in vivo administration of human chorionic gonadotropin and ethanol on the processes of testicular receptor depletion and replenishment

Abstract The effects of hCG (CR119) and ethanol (20% in saline, v/v) on the levels of gonadotropin receptor in mature rat testes are studied. The results indicate that 75 I.U. of hCG administered intraperitoneally is capable of depleting testicular tissue of all its gonadotropin binding sites within 24 hrs. after administration, and 11–14 days are required for these binding sites to be fully restored. The disappearance and reappearance patterns of testicular gonadotropin binding sites following in vivo ethanol administration are very similar to those seen after gonadotropin administration, the only difference being that ethanol is much less immediate in its effect. The level of hLH in the serum of these rats is unaffected by the ethanol administered which seems to indicate that reduction of the number of gonadotropin binding sites in testes brought about by ethanol is through membrane-mediated mechanism. For rats receiving gonadotropin injections, no correlation sseems to exist between the concentration of gonadotropin in serum and the level of detectable gonadotropin binding sites in the testis.

Gonadotropin receptor occupancy and stimulation of cAMP and testosterone production by purified Leydig cells: Critical dependence on cell concentration

Rat testicular interstitial cells have been separated by discontinuous/continuous gradient of Percoll, yielding four cell fractions. The light cells in fraction I bound luteinizing hormone/human chorionic gonadotropin (LH/hCG) with high affinity but were not steroidogenic in response to hormone. Fraction II consisted mainly of germ cells. Although fraction III contained Leydig cells, this fraction was contaminated with germ cells and was less responsive to hormone as compared to the Leydig cells in fraction IV. The Leydig cells in fraction IV produced cAMP and testosterone in response to hormone action in a manner which was critically dependent upon cell concentration. The production of cyclic adenosine monophosphate (cAMP) in the presence of saturating concentrations of hCG (2.4 X 10(-10) M) was linear as a function of cell concentration up to 7.0 X 10(6) cells/1.25 ml and thereafter, a slight inhibition (26%) was seen at 10 X 10(6) cells/1.25 ml. The average value for cAMP production by hCG was 133.8 +/- 8.5 pmol cAMP/2 X 10(6) cells. The production of testosterone was biphasic, increasing linearly up to 5 X 10(6) cells/1.25 ml and decreasing thereafter. Two million cells, in the presence of 2.4 X 10(-10) M hCG, produced an average of 24.2 +/- 1.7 ng of testosterone in reaction volumes ranging from 1 to 2 ml whereas the same number of cells only produced 5.1 +/- 0.6 ng of testosterone in 250 microliters. The binding of 125I-labeled hCG to the same batch of cells increased with increasing cell concentrations as expected but under the conditions of maximal steroidogenesis at low cell concentrations (1.25, 2.0, and 2.5 X 10(6) cells/1.25 ml), it was barely detectable. Thus, we conclude that there is an inverse relationship between the parameters of binding and biological response in purified Leydig cells.

Gonadal receptors II. Effect of time and reaction volume upon the binding of human chorionic gonadotropin and human luteinizing hormone to particulate receptors

The effect of reaction volume upon the binding of gonadotropins by particulate receptors was studied. Two experimental approaches were used: one involved increasing the reaction volume of the binding assay (i.e. diluting the hormone and receptor concentrations and will be referred to as buffer coincubation studies) and the other involved incubating the testicular homogenate in various buffer volumes prior to the binding assay (buffer preincubation stidies). The results showed that the number of hormone binding sites inferred from Scatchard analysis was inversely related to the reaction volume in the coincubation as well as in the preincubation studies. Time-dependent dissociation of receptors from the intact testis was demonstrated by perifusion studies and the loss of receptors from intact testis correlated with the appearance of soluble factors (Bhalla, V.K., Haskell, J., Grier, H. and Mahesh, V.B. (1976) J. Biol. Chem. 251, 4947--4957) in the eluate obtained. The results obtained along with those presented in the preceding manuscript (Chen, C.J.H., Lindeman, J.G., Trowbridge, C.G. and Bhalla, V.K. (1979) Biochim. Biophys. Acta 284, 407--435) question the validity of the rapid equilibrium model which assumes reversible hormone occupancy of a fixed number of receptor sites. An alternate binding model is proposed herein and its implications are discussed.

Hyperhomocysteinemia is detrimental to pregnancy in mice and is associated with preterm birth

Elevated levels of homocysteine produce detrimental effects in humans but its role in preterm birth is not known. Here we used a mouse model of hyperhomocysteinemia to examine the relevance of homocysteine to preterm birth. The mouse carries a heterozygous deletion of cystathionine β-synthase (Cbs(+/-)). Gestational period was monitored in wild type and Cbs(+/-) female mice. Mouse uterine and placental tissues, human primary trophoblast cells, and human myometrial and placental cell lines were used to determine the influence of homocysteine on expression of specific genes in vitro. The activity of BKCa channel in the myometrial cell line was monitored using the patch-clamp technique. We found that hyperhomocysteinemia had detrimental effects on pregnancy and induced preterm birth in mice. Homocysteine increased the expression of oxytocin receptor and Cox-2 as well as PGE2 production in uterus and placenta, and initiated premature uterine contraction. A Cox-2 inhibitor reversed these effects. Gpr109a, a receptor for niacin, induced Cox-2 in uterus. Homocysteine upregulated GPR109A and suppressed BKCa channel activity in human myometrial cells. Deletion of Gpr109a in Cbs(+/-) mice reversed premature birth. We conclude that hyperhomocysteinemia causes preterm birth in mice through upregulation of the Gpr109a/Cox-2/PGE2 axis and that pharmacological blockade of Gpr109a may have potential in prevention of preterm birth.

Is deglycosylated human chorionic gonadotropin an antagonist to human chorionic gonadotropin ? characterization of deglycosylated human chorionic gonadotropin action in two testicular interstitial cell fractions

In order to determine the significance of carbohydrate residues of human chorionic gonadotropin (hCG) in receptor interaction and signal transduction leading to steroidogenesis, the effect of deglycosylated hCG (DG-hCG) was studied in vitro with two different hCG-responsive purified testicular interstitial cell fractions. Fraction I light cells, previously found to bind 125I-labeled hCG with high affinity without producing testosterone, also bound 125I-labeled DG-hCG with high affinity (Kd 7.2.10(-10) M) without stimulating testosterone production. Fraction IV heavier cells, which produced testosterone in response to hCG without detectable high-affinity hCG-binding sites, neither bound DG-hCG nor sufficiently produced cAMP and testosterone in response. With the addition of intact hCG, DG-hCG inhibited cAMP levels, although not sufficiently to inhibit testosterone production. This observation was contrary to previous studies in which DG-hCG was shown to be an antagonist to hCG action. We conclude that: (a) DG-hCG retains its binding activity in light cells and this high-affinity binding is unrelated to steroidogenesis; (b) DG-hCG does not bind to heavier cells with high affinity and loses its biological activity as result of deglycosylation; (c) DG-hCG actions in this study strengthen the concept of two different hCG-responsive cells in the rat interstitium which, if not separated, will yield misleading data supporting the coexistence of hCG high-affinity binding and biological response in the same cell; and (d) DG-hCG partially antagonizes the activation of adenylate cyclase but does not block testosterone production, thus questioning the usefulness of this analogue in antagonizing the action of native hCG in rat testis.

Demonstration of hCG Binding Sites and hCG Stimulated Steroidogenesis in Different Populations of Interstitial Cells

The mechanism by which luteinizing hormone (LH) promotes the production of testosterone in Leydig cells by binding to its high affinity sites was reinvestigated. Collagenase dispersed interstitial cells when purified by the application of a variety of techniques such as unit gravity sedimentation, gradient centrifugation, and a combination of the two procedures, were separated into two LH/hCG responsive cell fractions. The two types of interstitial cells displayed distinct biochemical and morphological characteristics. One cell type (the light cell) bound 125I-labeled human chorionic gonadotropin (125I-labeled hCG) with high affinity (Ka approximately equal to 3.33 x 10(9) M-1) but testosterone was not produced by this cell type as a result of hCG target cell receptor interaction. On the other hand, hCG stimulated the production of testosterone in another cell type (the dark/heavier cell). Steroidogenesis was maximally stimulated (700-800 percent over basal) by concentrations of hCG in the range of 3 x 10(-10) M, but high affinity binding sites for 125I-labeled hCG were not detectable. The residual binding that occurred did not obey saturation kinetics and was predominantly nonspecific. The stimulation of steroidogenesis by hCG in dark/heavier cells was dose and time dependent. Addition of dibutyryl or bromo cAMP (1 mM) to the cell suspension resulted in production of testosterone demonstrating the involvement of an hCG sensitive adenylate cyclase system in the transfer signaling process. These observations suggest the lack of a direct association between the occupancy of high affinity binding sites by hCG and testosterone production in rat Leydig cells. The stimulation of a biological response by a pathway independent of hCG occupancy of high affinity binding sites on Leydig cell is discussed and morphology of light and dark/heavier cells is presented. Autoradiographic evidence substantiates the conclusions.

Apparent Lack of Involvement of cAMP as a Mediator of LHRH Stimulation of Nuclear Estrogen Receptor Activity in the Rat Anterior Pituitary

We have previously shown that incubation of rat pituitary cells in the presence of LHRH results in specific enhancement of nuclear estrogen receptor (ER) binding which cannot be accounted for by simple intracellular translocation of cytoplasmic receptor. In the present study, the role of cAMP in this response has been examined. Suspended pituitary cells from adult ovariectomized rats primed with estradiol were incubated with varying concentrations of LHRH or a highly active LHRH analog (LHRH-A) for 30 min at 37 degrees C, and levels of cAMP were determined. Total cAMP levels changed only in response to a concentration of 100 pmol/pituitary of either peptide; the stimulation by LHRH was twice that by LHRH-A. When the priming dose level of estradiol was reduced from 1.0 to 0.5 micrograms/day, stimulation of cells by 100 pmol of LHRH caused a much greater increase in cAMP levels. Separate incubation of subcellular fractions with a wide dose range of dibutyryl-cAMP (DBcAMP) resulted in a progressive loss of cytosol receptor binding capacity (which was also observed in whole cultured or suspended cells), but no significant concomitant change in nuclear receptor binding; however, whole cells in suspension or culture did show an increase in nuclear ER activity in the presence of 100 nM DBcAMP. This response was qualitatively, but not quantitatively, similar to that elicited by LHRH. When the effects of whole cell incubation with LHRH and LHRH-A on nuclear ER were compared with their effects on total cAMP levels, no correlation was observed; rising levels of cAMP did, however, coincide with falling levels of cytosol ER.(ABSTRACT TRUNCATED AT 250 WORDS)

Gonadotropin stimulation of cyclic adenosine monophosphate and testosterone production without detectable high-affinity binding sites in purified Leydig cells from rat testis

Rat testicular interstitial cells were separated by three different gradient-density procedures and, with each, two biochemically and morphologically distinct cell fractions were isolated. The lighter density cells in fraction-I bound iodine 125-labeled human chorionic gonadotropin (hCG) with high-affinity (apparent equilibrium dissociation constant, Kd, approximately 10(-10) M) without producing either cyclic adenosine monophosphate or testosterone in response to hormone action. The heavier-density cells displayed morphologic features typical of Leydig cells and produced cyclic adenosine monophosphate and testosterone in the presence of hCG without detectable 125I-labeled hCG high-affinity binding. These cell fractions were further characterized by studies using deglycosylated hCG, a known antagonist to hCG action. Cell concentration-dependent studies with purified Leydig cells revealed that maximal testosterone production was achieved when lower cell concentrations (0.5 x 10(6) cells/250 microliters) were used for in vitro hCG stimulation assays. Under these conditions, the 125I-labeled hCG binding was barely detectable (2.24 fmol; 2,698 sites/cell). Furthermore, these studies revealed that the hCG-specific binding in Leydig cells is overestimated by the classic method for nonspecific binding correction using excess unlabeled hormone. An alternate method is presented.

Studies on the nature of interaction between follicle-stimulating hormone and the testicular receptors

Two previous reports from this laboratory showed that the binding of 125I-labeled human choriogonadotropin and 125I-labeled human luteinizing hormone to rat testicular receptors is partially irreversible and the binding parameters obtained from Scatchard analysis of the data can be drastically altered simply by changing the reaction volume of the binding assays (Chen, C.J.H., Lindeman, J.G., Trowbridge, C.G. and Bhalla, V.K. (1979) Biochim, Biophys. Acta 584, 407--435; Bhalla, V.K., Trowbridge, C.G., Chen, C.J.H., Lindeman, J.G. and Rojas, F.J. (1979) Biochim, Biophys. Acta 584, 436--453). It is reported herein that the binding reaction between follicle-stimulating hormone and testicular receptors displays very similar characteristics. The results support the previous conclusion that receptor concentrations fluctuate in the membranes and that the extent of their loss from tissue membranes in vitro is dependent upon time and temperature of incubation, the volume of buffer present, and the quantity of hormone used.