Rat Testis Membranes Research Articles

Ribose-modified nucleosides as ligands for adenosine receptors: synthesis, conformational analysis, and biological evaluation of 1'-C-methyl adenosine analogues.

1'-C-Methyl analogues of adenosine and selective adenosine A(1) receptor agonists, such as N-[(1R)-1-methyl-2-phenylethyl]adenosine ((R)-PIA) and N(6)-cyclopentyladenosine, were synthesized to further investigate the subdomain that binds the ribose moiety. Binding affinities of these new compounds at A(1) and A(2A) receptors in rat brain membranes and at A(3) in rat testis membranes were determined and compared. It was found that the 1'-C-methyl modification in adenosine resulted in a decrease of affinity, particularly at A(1) and A(2A) receptors. When this modification was combined with N(6) substitutions with groups that induce high potency and selectivity at A(1) receptors, the high affinity was in part restored and the selectivity was increased. The most potent compound proved to be the 1'-C-methyl analogue of (R)-PIA with a K(i) of 23 nM for the displacement of [(3)H]CHA binding from rat brain A(1) receptors and a > 435-fold selectivity over A(2A) receptors. In functional assays, these compounds inhibited forskolin-stimulated adenylate cyclase with IC(50) values ranging from 0.065 to 3.4 microM, acting as full agonists. Conformational analysis based on vicinal protonminus signproton J-coupling constants and molecular mechanics calculations using the MM2 force field proved that the methyl group on C1' in adenosine has a pronounced impact on the furanose conformation by driving its conformational equilibrium toward the north, gamma+, syn form.

2'-C-Methyl analogues of selective adenosine receptor agonists: synthesis and binding studies.

2'-C-Methyl analogues of selective adenosine receptor agonists such as (R)-PIA, CPA, CCPA, NECA, and IB-MECA were synthesized in order to further investigate the subdomain that binds the ribose moiety. Binding affinities of these new compounds at A1 and A2A receptors in bovine brain membranes and at A3 in rat testis membranes were determined and compared. It was found that the 2'-C-methyl modification resulted in a decrease of the affinity, particularly at A2A and A3 receptors. When such modification was combined with N6-substitutions with groups which induce high potency and selectivity at A1 receptors, the high affinity was retained and the selectivity was increased. Thus, 2-chloro-2'-C-methyl-N6-cyclopentyladenosine (2'-Me-CCPA), which displayed a Ki value of 1.8 nM at A1 receptors, was selective for A1 vs A2A and A3 receptors by 2166- and 2777-fold, respectively, resulting in one of the most potent and A1-selective agonists so far known. In functional assay, this compound inhibited forskolin-stimulated adenylyl cyclase activity with an IC50 value of 13.1 nM, acting as a full agonist.

RU 3117 a steroidal compound with high affinity for sigma sites in rat testis membranes.

RU 3117 belongs to a new series of steroids which exhibited a high relative binding affinity (RBA) for (+)[3H]PPP sites in rat testis membranes; its RBA was about 40 times higher than that of progesterone. Furthermore, it is devoid of any binding to classical steroid receptors; therefore in order to study its binding parameters on rat testis membranes it was tritiated. [3H]RU 3117 bound at least two distinct sites with Ka values of 0.4 +/- 0.06 x 10(9) M(-1) and 1.3 +/- 0.2 x 10(7) M(-1). Using this marker, competition studies with cold haloperidol showed that a part of this binding was haloperidol-sensitive, whereas another part was haloperidol-resistant. Interestingly, progesterone described as a sigma ligand competes with [3H]RU 3117 binding, with an RBA of 1.6%. When haloperidol was preincubated (250 nM) with rat testis membranes, in order to mask the sigma sites, we observed that DTG (1,3-di-O-tolylguanidine) and haloperidol displayed a very low RBA (< 0.1%) and were not able totally to displace the [3H]RU 3117 binding up to 50 microM. Furthermore, benztropine exhibited a significant RBA of 19% but its displacement curve showed a plateau (500-50,000 nM). These results showed that part of the haloperidol-resistant sites was benztropine sensitive but another part was displaced neither by haloperidol nor by benztropine. The presence of these remaining binding sites was confirmed by preincubating a mixture of haloperidol and benztropine with testis membranes. Under these conditions, [3H]RU 3117 displayed a Ka of 1.0 +/- 0.01 x 10(7) M(-1), and we observed that these sites were recognized, up to now, only by the steroids RU 1968 and RU 54173 which are also devoid of any binding to classical nuclear steroid receptors.

Synthetic peptides corresponding to residues 551 to 555 and 650 to 653 of the rat testicular follicle-stimulating hormone (FSH) receptor are sufficient for post-receptor modulation of Sertoli cell responsiveness to FSH stimulation

We have recently demonstrated that synthetic peptides corresponding to the third cytoplasmic (3i) loop (residues 533 to 555) and a region in the carboxy-terminal cytoplasmic tail (residues 645 to 653) of the rat testicular follicle-stimulating hormone receptor (FSHR) affected signal transduction in rat testis membranes and cultured rat Sertoli cells. In order to define more precisely the peptide domains involved, we synthesized truncated peptide amides corresponding to FSHR residues 551–555 (KIAKR) and 650–653 (RKSH), respectively. These two regions were chosen since they contained a minimal structural motif present in G protein activator regions of several other G protein-coupled receptors (i.e., B-X-X-B-B or B-B-X-B, B representing a basic amino acid). Neither peptide inhibited binding of FSH to testis membrane receptors. Each peptide significantly reduced FSH-stimulated estradiol biosynthesis by intact cultured rat Sertoli cells. The same results were obtained with streptolysin O-permeabilized Sertoli cells. No effect was noted on forskolin-induced steroidogenesis, indicating that the peptide effects were not due to interaction with adenylyl cyclase. Each peptide amide, however, induced concentration-dependent increases in guanine nucleotide exchange in rat testis membranes. Our results indicate that interaction of FSH receptor with its associated G protein may involve relatively restricted peptide sequences, and include residues 551–555 (KIAKR) in the third cytoplasmic loop, and residues 650–653 (RKSH) in the carboxy-terminal cytoplasmic tail of the FSH receptor.

A synthetic peptide corresponding to the third cytoplasmic loop (residues 533 to 555) of the testicular follicle-stimulating hormone receptor affects signal transduction in rat testis membranes and in intact cultured rat Sertoli cells

Involvement of the third cytoplasmic (3i) loop (residues 533 to 555) of the rat testicular FSH receptor in the mechanism of FSH signal transduction was examined using light membranes prepared from immature rat testes, monolayer cultures of rat Sertoli cells, and a synthetic peptide strategy. This region of the FSH receptor is structurally related to G protein-activator regions identified in other G protein-coupled receptors. FSHR-(533–555) peptide amide stimulated guanine nucleotide exchange in rat testis light membranes, presumably via its interaction with membrane-associated G protein. The peptide failed to inhibit FSH binding to testis membrane receptors, indicating that the nucleotide exchange effect was not a result of peptide interaction with receptor. When incubated with cultured Sertoli cells from immature rat testes, FSHR-(533–555) peptide amide consistently and significantly inhibited FSH stimulation of cAMP and estradiol biosynthesis, but failed to inhibit forskolin stimulation of each. The peptide effect, therefore, was not due to a direct interaction with adenylyl cyclase. Since FSHR-(533–555) peptide amide did not inhibit FSH binding to membrane receptor, these results imply entry of the peptide into the Sertoli cell, possibly by vesicular internalization or diffusion. Indeed, the inhibitory effects of FSHR-(533–555) peptide amide on FSH-stimulated estradiol biosynthesis were prevented by pretreating Sertoli cells with phenylarsine oxide, an inhibitor of FSH receptor internalization. FSHR-(533–555) was without effect on basal levels of cAMP and estradiol biosynthesis, indicating absence of toxicity at the concentrations tested. Synthetic peptide amides lacking structural criteria associated with G protein activation did not affect guanine nucleotide exchange or FSH-stimulated estradiol levels, even at concentrations significantly higher than used for the receptor-related peptide. Our results are consistent with peptide interaction with signal-transducing G protein. These data suggest that the 3i loop peptide acts as an antagonist of FSH receptor-mediated G protein activation, and inhibits FSH receptor-mediated signal transduction in intact Sertoli cells. The ability of this FSH receptor-related peptide to affect intracellular processes in intact cultured rat Sertoli cells through its apparent interaction with G protein suggests a novel approach for control of FSH action.

A synthetic peptide corresponding to residues 645–653 in the carboxyl terminal cytoplasmic domain of the rat testicular follicle stimulating hormone receptor modulates G protein coupled-receptor signaling in rat testis membranes and in intact cultured rat Sertoli cells

We examined the involvement of the carboxyl terminal cytoplasmic domain (residues 645–653) of the rat testicular FSH receptor in FSH signal transduction utilizing light membranes prepared from immature rat testes, intact cultured rat Sertoli cells, and a synthetic peptide approach. This region of the FSH receptor was selected because of its structural similarity to receptor-G protein contact sites identified in other G protein-coupled receptors. FSHR- (645–653) peptide amide promoted guanine nucleotide exchange in rat testis membranes, presumably via its interaction with membrane-associated G protein, but did not inhibit FSH binding to testis membrane receptors. When incubated with intact cultured Sertoli cells from immature rat testes, FSHR-((645–653) peptide amide consistently and significantly stimulated basal cAMP and estradiol biosynthesis. The peptide had no effect on forskolin stimulation of cAMP and estradiol, but inhibited FSH stimulation of each. Since FSH binding to receptor was unaffected by the peptide, these results suggest peptide interaction with receptor-associated G protein. The effects of FSHR- (645–653) peptide amide on FSH-stimulated estradiol biosynthesis were prevented by pretreating Sertoli cells with phenylarsine oxide, an inhibitor of FSH receptor internalization. These results suggest that peptide effects in intact Sertoli cells were related to peptide entry into the cell, presumably during receptor-mediated endocytosis of FSH, or by diffusion. Synthetic peptide amides not satisfying structural criteria for G protein coupling had no effect on either guanine nucleotide exchange or estradiol biosynthesis, even at concentrations significantly higher than used for FSHR- (645–653) peptide amide. Although the mechanisms involved in the effects of FSHR- (645–653) peptide amide on FSH-stimulated postbinding events are not completely understood, our results are consistent with interaction of the peptide with signal-transducing G protein. The results of this study further suggest that modulation of hormone action by effects on G protein-related events in intact cells is feasible, and that this intervention may offer an approach for control of FSH action at a level other than receptor binding.

Characterization of a low affinity binding site for N6-substituted adenosine derivatives in rat testis membranes.

The binding characteristics of radiolabeled N6-(cyclohexyl)adenosine ([3H]CHA), N6-(R-phenylisopropyl)adenosine ([3H]R-PIA), 5'-N-ethyl-carboxamidoadenosine ([3H]NECA), and 2-[4-(2-carboxyethyl)phenyl]ethyl-amino-5'-N-ethylcarboxamidoadenosine ([3H]CGS 21680), to rat testis membranes were investigated. Specific binding of [3H]CGS 21680, a selective agonist for the A2a adenosine receptor, was very modest whilst the nonselective agonist [3H]NECA bound to rat testis membranes showing high binding capacity. At least two types of binding sites for [3H]NECA could be identified in rat testis membranes: high affinity sites and high capacity sites. Selective agonists for the A1 adenosine receptor, [3H]CHA and [3H]R-PIA bound with high affinity to a single class of binding sites. This high affinity binding site showed the typical pharmacological specificity of the A1 adenosine receptor with a potency order for agonists of CHA > or = R-PIA > NECA > N6-(S-phenylisopropyl)adenosine (S-PIA). In order to detect the presence of the A3 adenosine receptor in these membranes we selectively blocked the A1 receptor with a large molar excess of a xanthine antagonist, either 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) or xanthine amine congener (XAC). In the presence of an antagonist a low affinity binding site for [3H]CHA and [3H]R-PIA was detected. This low affinity binding site showed a different pharmacological specificity than the high affinity binding site. In fact the potency order for agonists was CHA > or = NECA = R-PIA > S-PIA. This finding suggests that the low affinity binding site represents the A3 adenosine receptor.

Site-directed alanine mutagenesis of Phe33, Arg35, and Arg42-Ser43-Lys44 in the human gonadotropin alpha-subunit.

Residues Phe33 and Arg35, individually, and a composite mutation of residues Arg42, Ser43, and Lys44 were changed to alanine in the human glycoprotein hormone common alpha-subunit using site-directed mutagenesis. These specific residues are highly conserved across species and have by chemical modification and synthetic peptide approaches been implicated in the binding of human chorionic gonadotropin (hCG) to leutinizing hormone (LH) receptor. In the present study we tested the hypothesis that specific alpha-subunit amino acid residues which stabilize the hormone receptor interaction for hCG have the same function in human follicle-stimulating hormone (hFSH). Wild type or mutant alpha-subunit cDNAs were coexpressed with wild type hFSH or hCG beta cDNA in sialylation defective Chinese hamster ovary cells. Recombinant hormones were tested in a radioligand receptor competition assay, using rat testis membranes as a source of FSH and LH receptors. Mutant hFSH heterodimers F33A-FSH, R35A-FSH, Arg42-Ser43-Lys44/Ala42-Ala43-Ala44- FSH all displaced 125I-hFSH in a similar fashion, indicating that these residues are not important for binding of hFSH to the rat FSH receptor. On the other hand, F33A-CG evidenced a 5-fold decrease in binding, while R35A-CG had over a 100-fold decrease in binding to the rat LH receptor when compared to the wild type recombinant hCG. These data demonstrate that a receptor-binding site on the common alpha-subunit which is very important for hCG binding to LH receptor is not important for the binding of hFSH to FSH receptor. Our interpretation of these findings is that there are fundamental structural differences in the receptor interface contacts of the common alpha-subunit, which stabilize receptor binding among members of the glycoprotein hormone family.

In Vitroandin VivoBioactivity of Recombinant Human Follicle-Stimulating Hormone and Partially Deglycosylated Variants Secreted by Transfected Eukaryotic Cell Lines*

Recent studies have shown that Chinese hamster ovary (CHO) cells transfected with the FSH subunit genes secrete bioactive FSH. Here, we determined the in vitro and in vivo bioactivity of recombinant FSH produced by CHO mutant cells deficient in the glycosylation enzyme N-acetylglucosamine transferase-I (NAGT-), resulting in glycoproteins with asparagine-linked (GlcNAc)2(Mannose)5 oligosaccharides, or mutant cells defective in sialic acid transport into the Golgi (ST-). In the latter, glycoproteins are secreted lacking terminal sialic acids. Determination of in vitro bioactivity, using the granulosa cell aromatase bioassay, indicated that both FSH variants are as active as FSH secreted by the wild type (WT) cells and purified pituitary FSH. Also, these normal and variant forms of FSH are equipotent in a radioligand receptor assay using rat testis membranes. However, the variant FSH molecules are more basic than the WT FSH as determined using a chromatofocusing column (pI: wild type 3.6-5.0, NAGT- greater than 7.0, ST- approximately 6.0 and greater than 7.0). Injection of immature estrogen-treated rats with WT FSH induced high aromatase activity in their granulosa cells whereas treatment with either one of the FSH variants was ineffective; the lack of in vivo activity of the FSH variants was correlated with rapid clearance of these molecules in serum. Thus, recombinant human FSH produced by cells deficient in NAGT-I or defective in sialic acid transport retains normal receptor binding and in vitro bioactivity, but exhibits minimal in vivo activity and a shortened half-life when compared to WT FSH, indicating the important role of terminal sugars for FSH action in vivo.

A1 adenosine receptor of rat testis membranes. Purification and partial characterization.

Purification of an A1 adenosine receptor of rat testes was performed using a newly developed affinity chromatography system (Nakata, H. (1989) Mol. Pharmacol. 35, 780-786). The A1 adenosine receptor was solubilized with digitonin from rat testicular membranes and then purified more than 25,000-fold by sequential use of affinity chromatography on xanthine amine congener-immobilized agarose, hydroxylapatite chromatography, re-affinity chromatography on xanthine amine congener-agarose, and finally gel permeation chromatography on TSK-3000SW. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the final preparation showed a single broad band of Mr 41,000 by autoradiography after radioiodination. This Mr 41,000 peptide was also specifically labeled with an A1 adenosine receptor affinity labeling reagent. A high affinity A1 adenosine receptor antagonist, 8-cyclopentyl-1,3-[3H]dipropylxanthine, bound saturably to the purified receptor with a KD of approximately 1.4 nM. The purified receptor also showed essentially the same specificity for adenosine agonists and antagonists as the unpurified receptor preparations, although the affinities of the purified adenosine receptor for agonists were significantly low compared to those of unpurified receptor preparations indicating that the purified A1 adenosine receptor exists as a low agonist-high antagonist affinity state. Deglycosylation of the purified testis adenosine A1 receptors with endoglycosidase F produced an increase in the mobility of the receptor protein to an apparent Mr 30,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, similar to that of deglycosylated A1 adenosine receptors of rat brain membranes. Peptide maps of the purified testis and brain A1 adenosine receptors using trypsin and V8 protease suggest that these receptors show some structural homologies.

Characterization of high-density lipoprotein binding sites in rat liver and testis membranes

The binding of human 125I-labeled HDL 3, to purified rat liver and testis plasma membranes was studied. About 50–65% of the total HDL binding in these membranes was abolished by 1% bovine serum albumin in the incubation medium. The remaining albumin-insensitive binding sites, determined in the presence of albumin were associated with plasma membranes; a good correlation was found between the 125I-labeled HDL 3 binding and the 5'-nucleotidase activities of the membrane fractions. The binding sites in these tissues were saturable, specific for HDL (not competed for by LDL) and had similar affinities for 125I-labeled HDL 3 ( K d , 11.8 μg protein/ml for liver and 12.7 μg protein/ml for testis membranes); the maximum binding capacity of the testis membranes was higher (1.3 vs. 0.7 μg protein/mg membrane protein). Egg phosphatidylcholine complexes of both human apolipoprotein A-I and apolipoprotein C's competed for the HDL-binding sites, but phosphatidylcholine vesicles alone did not. Chemical modification of the lysine and arginine residues of apolipoproteins did not affect the interaction of HDL 3 with its binding sites. Despite the fact that the HDL-binding sites in these tissues are not specific for apolipoprotein A-I, they may have important physiological roles in lipid transport, as they appear to recognize apolipoprotein-phospholipid complexes.

Inhibition by certain plant extracts of the binding and adenylate cyclase stimulatory effect of bovine thyrotropin in human thyroid membranes.

The present studies were undertaken to explore the mechanism by which, as previous studies have shown, freeze-dried aqueous extracts (FDE) of plants of the species Lycopus virginicus and Lycopus europaeus, Melissa officinalis (Laminaceae), and Lithospermum officinale (Boraginaceae) have the ability to inhibit at least many of the effects of exogenous and endogenous TSH on the thyroid gland. To this end, we have examined the in vitro effects of FDE from these plants on the ability of bovine TSH (bTSH) to both bind to human thyroid plasma membranes (TPM) and activate adenylate cyclase therein. FDE of these four species produced a dose-related, ultimately complete, inhibition of the binding of 125I-labeled bTSH when studied at 4 C in a 20 mM Tris-HCl-0.5% BSA buffer, pH 7.45. Half-maximum inhibition of bTSH binding was produced by approximately 50 mU/ml bTSH and only about 10-30 micrograms/ml of the four active FDE. When studied in Tris-BSA-50 mM NaCl buffer at 37 C, these FDE remained inhibitory to bTSH binding, but their potency was decreased to about one fifth of that seen in the absence of NaCl. The binding of [125I]hCG to rat testis membranes was also inhibited by all of these FDE, but no effect on the binding of [125I]insulin to crude rat liver membranes was observed. In concentrations as high as 1 mg/ml, FDE of Verbena officinalis (Verbenaceae), which belongs to the same order (Tubiflorae) as the other plants, but exhibits no antithyrotropic or antigonadotropic activity in vivo, had no effect on either the binding of bTSH to thyroid membranes or the binding of hCG to rat testis membranes. No inhibition of [125I]bTSH binding occurred when TPM were preincubated with the four active FDE, washed, and then incubated with [125I]bTSH in medium devoid of FDE. Hence, the inhibition of [125I]bTSH binding seen when labeled hormone and active FDE were added together was not due to irreversible binding of FDE to TPM or damage to the TSH receptor. When [125I]bTSH was incubated with the active FDE in Tris-BSA and the mixture was chromatographed on Sephadex G-100 using the same buffer, [125I]bTSH was shifted from an apparent mol wt of 30,000 and eluted at the void volume. Direct binding of [125I]bTSH in fractions from the new, large molecular peak was nil. Addition of a large excess of unlabeled bTSH during preincubation prevented the shift in the elution pattern of [125I] bTSH produced by these FDE.(ABSTRACT TRUNCATED AT 400 WORDS)

Changes in insulin binding in rat testis following testosterone and gonadotrophins administration.

Rat testis membranes possess insulin receptor whose autoregulation is different from conventional insulin target organs. In order to verify the importance of the functional activity of the Leydig cells, different approaches were selected to provoke a resting state of the cells and to measure insulin binding. Testosterone cypionate and LH-RH agonist administration, as well as hypophysectomy, led to a 50% decrease of insulin binding in testis while no effect or a slight increase was observed in hepatic membranes following hypophysectomy. HCG treatments restored insulin binding to its control levels in hypophysectomized animals. These modifications occurred without significant changes in insulin and glucose concentrations. It is concluded that pituitary LH is a key factor in controlling Leydig cell activity as well as its insulin receptor content.

The structure and function of glycoprotein hormone receptors: Ganglioside interactions with luteinizing hormone

Summary Gangliosides inhibit the binding of 125 I-labeled luteinizing hormone to rat testis membranes. The inhibition is the result of an interaction between the hormone and the ganglioside rather than the membrane and ganglioside, and the interaction with ganglioside can be detected by fluorescence spectroscopy. In both the binding inhibition and fluorescence studies, luteinizing hormone recognizes an oligosaccharide sequence on the ganglioside molecule distinct from that of thyrotropin, human chorionic gonadotropin, and cholera toxin.