Factors regulating the response of cells cultured from human giant cell tumors of bone to parathyroid hormone

Exposure of cells cultured from human giant cell tumors of bone to parathyroid hormone (PTH) results in an increase in the intracellular cAMP content. When cells are preincubated with PTH for periods as short as 10 min and then reexposed to this hormone, there is a decrease in the subsequent PTH-induced cAMP response. This decrease is inversely related to the concentration of PTH present during the preincubation. To investigate the factors responsible for this decrease in hormone response, we used a synthetic substituted analog of bovine PTH, [Nle8, Nle18, Tyr34]bPTH-(3-34) amide (PTH-Inh), which had previously been shown to be a competitive inhibitor of PTH-induced cAMP response in renal and bone-derived tissues. Incubation of these cultured cells with this analog failed to increase intracellular cAMP levels. In contrast to the results obtained with native hormone, when cells were preincubated with PTH-Inh, the acute cAMP response to native PTH after the removal of PTH-Inh was not diminished. In addition, simultaneous preincubation with native PTH and PTH-Inh partially overcame desensitization to PTH. These results suggest that occupancy of the PTH receptor alone is not sufficient to cause desensitization. Rather, steps subsequent to hormone binding, including activation of adenylate cyclase, may be required for the induction of a state of desensitization to PTH.     

Mechanisms of desensitization to parathyroid hormone in human osteoblast-like SaOS-2 cells.

Signal transduction by the PTH receptor is now known to involve generation of multiple second messengers. Desensitization of the adenylate cyclase response to PTH is a common feature of bone- and kidney-derived target cells; however, no single mechanism appears to explain desensitization in the different cell types studied. To examine the role of protein kinase-A (PKA) in homologous desensitization to PTH, we employed human SaOS-2 osteoblast-like cells and a mutant subclone (Ca 4A), which expresses an inducible cAMP-resistant form of PKA. Pretreatment of SaOS-2 cells with PTH for 4 h reduced by 60-80% the cAMP response to subsequent rechallenge with the hormone. This homologous desensitization was significantly, but not completely, inhibited in Ca 4A cells. Desensitization was not mimicked by pretreatment of the cells with forskolin. PTH binding to its receptor was reduced 50% in both SaOS-2 and Ca 4A cells after 4-h incubation with PTH (homologous down-regulation), whereas forskolin did not cause receptor down-regulation. Pretreatment with the ionophore ionomycin for 4-24 h did not mimic desensitization to PTH. Both desensitization to PTH and receptor down-regulation were induced, however, by pretreatment with a phorbol ester (12-O-tetradecanoyl phorbol-13-acetate), and these effects were blocked completely by staurosporine. PTH-induced desensitization was not blocked by staurosporine, and receptor down-regulation was enhanced by the drug. Pertussis toxin did not prevent desensitization induced by either PTH or 12-O-tetradecanoyl phorbol-13-acetate. We conclude that homologous desensitization to PTH in SaOS-2 cells involves both cAMP-dependent and -independent mechanisms. Homologous PTH receptor down-regulation apparently is mediated by mechanisms independent of PKA activation. Neither pathway of homologous desensitization to PTH involves the action of pertussis toxin-sensitive G-proteins.     

Desensitization of LLC-PK1 cells by vasopressin results in receptor down-regulation

The molecular mechanism of desensitization of antidiuretic hormone receptors is not well understood. Preincubation of LLC-PK₁ cells with lysine vasopressin (LVP) (10⁻⁶ M, 5 h) decreased subsequent LVP-stimulated cAMP accumulation in cells by 83% and reduced the V_max of LVP-stimulated adenylate cyclase by 81%. Such preincubation also reduced by 90% the binding of [³H]LVP to both intact cells and isolated plasma membranes, suggesting a loss of vasopressin receptors. Both the reduction in cAMP response and the apparent loss of receptors showed similar dose and time dependence. Monensin (33 μM) did not alter [³H]LVP binding or stimulation of cAMP by LVP, nor did it prevent desensitization. However, membranes prepared from cells preincubated with LVP in the presence of monensin did not show a decrease in [³H]LVP binding. Forskolin preincubation, at 0.1, 1, 10 and 100 μM, did not alter [³H]LVP binding or accumulation of cellular cAMP by LVP, nor did it induce desensitization to LVP. Cells desensitized with varying LVP concentrations in the presence of 10 μM forskolin displayed the same loss of [³H]LVP binding and LVP responsiveness as observed in the absence of forskolin. LVP-desensitized cells, upon removal from LVP-containing medium, recovered cAMP responsiveness to LVP and specific binding of [³H]LVP at the same rate, achieving control levels after 50 h. Recovery was prevented by cycloheximide (25 μg/ml). These findings are consistent with a desensitization process involving LVPmediated receptor internalization, and a recovery process requiring protein synthesis.     

Protein kinase C is involved in PTH-induced homologous desensitization by directly affecting PTH receptor in the osteoblastic osteosarcoma cells.

We have investigated mechanisms of PTH-induced homologous desensitization reflected in the refractoriness of cAMP response to the second exposure to PTH in the clonal rat osteosarcoma cell line, UMR-106. Preincubation with 10(-7) M rat (r) PTH-(1-34) for 6 h caused the desensitization, resulting in a 65% decrease in cAMP accumulation in response to further exposure to rPTH. This desensitization was apparent at 10(-10) M rPTH and maximal at 10(-7) M rPTH. UMR-106 cells treated with protein kinase C (PK-C) activating phorbol ester, phorbol 12-myristate 13-acetate (PMA, 10(-6) M) for 6 h also induced desensitization manifested by a loss of rPTH-stimulated cAMP accumulation to 50% of that in the control cells. On the other hand, 4 alpha-phorbol 12,13-didecanoate, incapable of activating PK-C, failed to induce desensitization. Fifty micromolar H-7 (PK-C inhibitor) significantly blocked both rPTH- and PMA-induced desensitization. Thus, PK-C seemed to play a major role in rPTH-induced desensitization. Pretreatment with neither rPTH nor PMA changed the cAMP responsiveness to 10 micrograms/ml cholera toxin or 100 microM forskolin. Islet activating protein failed to influence the desensitization in this cell line. PTH receptor binding, assessed by using 125I-labeled [Nle8,Nle18,Tyr34]PTH-(1-34) as a radioligand, was decreased along with PTH receptor numbers by pretreatment with rPTH or PMA. These data indicate that rPTH-induced homologous desensitization occurs at least in part through the activation of PK-C and that PK-C directly affects PTH receptor in UMR-106 cells.     

Thyroid-stimulating antibody mimics thyrotropin in its ability to desensitize the adenosine 3',5'-monophosphate response to acute stimulation in continuously cultured rat thyroid cells (FRT-L5)

TSH preincubation induces refractoriness to further stimulation of cAMP accumulation by the hormone in cultured thyroid cells. The question of whether the thyroid-stimulating antibodies (TSAb) present in sera of patients with Graves' disease have the same desensitizing effect as TSH was evaluated in this study using a differentiated strain of rat thyroid cells (FRT-L5) which requires TSH for growth and develops refractoriness to acute TSH stimulation of cAMP accumulation. Cells cultured for 4-5 days in medium deprived of TSH recovered responsiveness to TSH and TSAb. Refractoriness to TSH was reinduced in these cells by a 24-h preincubation with 250 microU/ml TSH. The addition of 100 microM KI or 100 microM methimazole to the medium together with TSH did not modify TSH-induced refractoriness. Four different TSAb preparations all induced refractoriness to TSH-stimulated cAMP accumulation. TSAb preincubation induced refractoriness to acute TSAb as well as TSH-stimulated cAMP accumulation. TSAb-induced desensitization was dose dependent and required prolonged (8-24 h) exposure to TSAb. Furthermore, the desensitization induced by TSAb as well as that caused by TSH were significantly inhibited by cycloheximide, indicating the need for de novo protein synthesis. In conclusion, TSAb mimics TSH in the induction of refractoriness in FRT-L5 cells.     

Parathyroid hormone modulation of 25-hydroxyvitamin D3 metabolism by cultured chick kidney cells is mimicked and enhanced by forskolin

In order to determine whether cAMP mediates the effects of PTH on the metabolism of 25-hydroxyvitamin D3 (25-OH-D3) on chick kidney cells in primary culture, the effect of forskolin on the production of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] was assessed. In 4-h incubations with [3H]25-OH-D3 and forskolin, (1-10 microM) [3H]1,25-(OH)2D3 accumulation was increased 50-100%, and that of [3H]24,25-(OH)2D3 was decreased 30-60%. PTH (1-10 ng/ml) brought about identical changes. Similar results were observed when cultures were preincubated with nonradioactive 25-OH-D3 for 4 h in the presence of PTH and forskolin, followed by a 30-min incubation with radioactive substrate. At a low concentration (0.05 microM), forskolin alone had no effect on the metabolism of [3H]25-OH-D3 but markedly enhanced that of PTH. At maximal concentrations of PTH (10 ng/ml) and forskolin (10 microM), the effects of the two on 25-OH-D3 metabolism were not additive. Both PTH and forskolin decreased the further metabolism of [3H]1,25-(OH)2D3, probably by inhibiting its 24-hydroxylation, but there are also cycloheximide-sensitive steps in the metabolism of 1,25-(OH)2D3 that are not affected by PTH and forskolin. In time course experiments, increased [3H]1,25-(OH)2D3 accumulation could be observed before the detection of 24-hydroxylase activity suggesting that the primary effect of PTH and forskolin is on the production of [3H] 1,25-(OH)2D3 rather than its catabolism. Raising the calcium concentration of the medium to 2.5 mM from the normal 1.8 mM or lowering it to 0.5 mM for 24 h in serum-free medium did not alter the response of 25-OH-D3 metabolism to these agents. The results of these studies indicate that the effects of PTH on the metabolism of 25-OH-D3 by chick kidney cells are mediated by cAMP, since they can be enhanced and mimicked by forskolin, that they are exerted at the level of both 1- and 24-hydroxylase activity, and that they are not dependent on the calcium concentration of the medium.     

Response of adrenal cells to parathyroid hormone stimulation

Dispersed adrenal cells of avian and bovine origin were incubated with human (h) 1-34 parathyroid hormone (PTH(1-34)), bovine (b) PTH(1-84), bPTH(3-34), avian (a) PTH, and with ACTH. Kidney tubular cells, the established target cells for PTH, were used for comparison. Cyclic AMP (cAMP) accumulation and steroid hormone (aldosterone and corticosterone) secretion were measured in response to the hormones. In the bovine adrenocortical cells PTH of both bovine and avian origin stimulated cAMP production, but the aPTH action was more pronounced. The maximal cAMP response of avian adrenocortical cells to aPTH was 15-fold greater than that of ACTH, but a 20-times higher concentration of aPTH was required to reach half-maximal response. Avian PTH stimulated steroid hormone secretion in the chick adrenocortical cells, but the induced secretion was similar to that induced by ACTH, despite the difference in cAMP accumulation. Human PTH(1-34) and bPTH(1-84), which stimulated cAMP production in kidney cells, and the conventional antagonist bPTH(3-34) inhibited aPTH stimulation of cAMP accumulation in avian adrenocortical cells, but did not interfere with ACTH action. Furthermore, cAMP stimulation by aPTH and ACTH in avian adrenal cells was additive. The results establish the adrenal as a target organ for PTH, and suggest that the PTH acts through specific receptors, distinct from those for ACTH.     

Forskolin-induced homologous desensitization via an adenosine 3',5'-monophosphate-dependent mechanism(s) in human osteoblast-like SaOS-2 cells.

We have reported that pretreatment of human SaOS-2 osteoblast-like cells with forskolin (Fsk; 10(-5) M) for 4 h strikingly inhibited subsequent cAMP responsiveness to a second challenge with Fsk (Fsk-induced homologous desensitization) without altering the responses to PTH or vasoactive intestinal peptide (VIP). Pretreatment with PTH acutely augmented Fsk responsiveness, despite desensitizing the cells to rechallenge with PTH. The present studies were performed to investigate the mechanism of this differential desensitization. Fsk-induced desensitization was not mimicked by 1,9-dideoxyforskolin, a Fsk analog that does not activate adenylate cyclase (AC) but does reproduce certain cAMP-independent effects of Fsk. Fsk-induced homologous desensitization was also completely blocked in a cAMP-resistant mutant SaOS-2 cell line (Ca 4A), in which protein kinase-A (PKA) is not activated by endogenous cAMP. However, pretreatment with PTH (or VIP), which induced a large increase in cAMP, did not attenuate, but, rather, increased, the subsequent cAMP response to Fsk. Potentiation by PTH was also observed in Ca 4A cells. Pretreatment of SaOS-2 cells with pertussis toxin (100 ng/ml) for 12 h strikingly inhibited the initial cAMP response to Fsk, although Fsk-induced homologous desensitization was still clearly observed. Pretreatment with cholera toxin (1 microgram/ml) completely prevented Fsk-induced homologous desensitization. Combinations of maximal concentrations of Fsk plus hormones such as human PTH, human PTH-related peptide, or VIP elicited cAMP responses that were much more than additive, an effect not observed with combinations of hormones alone. We conclude that 1) Fsk-induced homologous desensitization of the AC response of SaOS-2 cells to a second challenge with Fsk is dependent upon activation of PKA; 2) one or more pertussis toxin-sensitive G-proteins contribute to full AC activation by Fsk, but are not involved in homologous desensitization; 3) augmentation by PTH (or VIP) pretreatment of Fsk-dependent AC activation involves an effector(s) other than PKA. These results provide further evidence that the regulation of AC responsiveness in SaOS-2 cells by PTH or VIP is complex and cannot be explained by activation of PKA alone.     

Formation of parathormone 8-34 by cathepsin-D digestion of parathormone and its efficacy as a hormone antagonist

It previously has been shown that digestion of bovine parathormone (bPTH) with cathepsin-D results in rapid cleavage of the hormone between Phe34 and Val35 yielding PTH(1-34) and PTH(35-84). Since bPTH also contains a Phe at residue 7 we have conducted additional studies to determine whether cleavage at this position could occur. We have found that following longer incubation periods of hormone and enzyme, 2 additional peptides are generated; PTH(8-34) and PTH(1-7). Time course studies demonstrated that these 2 fragments are formed from the (1-34) peptide generated through the initial cleavage at Phe34-Val35 of PTH. The identification of the bPTH(8-34) was accomplished through amino acid analysis and N-terminal sequencing. bPTH(8-34) behaved as a PTH antagonist in an in vitro mouse calvarial bone resorption assay. Although bPTH(8-34) did not affect the PTH-stimulated cAMP response when added simultaneously with PTH, preincubation of bone cells with this peptide caused desensitization of the PTH-stimulated cAMP response.     

Homologous and heterologous control of bone cell adenosine 3',5'-monophosphate response to hormones by parathoromone, prostaglandin E2, calcitonin, and 1,25-dihydroxycholecalciferol

Desensitization of hormone-stimulated cAMP response to parathormone, prostaglandin E2, and calcitonin was characterized in isolated bone cells. Osteoclast- and osteoblast-like cells derived from mouse calvaria were incubated for up to 24 h with agents that cause bone resorption (parathormone, prostaglandin E2, or 1,25-dihydroxycholecalciferol) or with calcitonin, which inhibits bone resorption. The cells were then exposed to parathormone, prostaglandin E2, or calcitonin, and cAMP formation was quantified. Homologous desensitization (refractoriness to the second exposure to the same hormone) occurred for each hormone and was essentially complete after 30 min of preincubation. Heterologous desensitization to parathormone could be produced in both cell types by 1,25-dihydroxycholecalciferol and prostaglandin E2, but not by calcitonin. Heterologous desensitization was of lesser magnitude, requiring more than 8 h to reach significant proportions. In contrast, the cAMP response to calcitonin and prostaglandin E2 could not be altered by preincubation with any other hormone. Dibutyryl cAMP or 3-isobutyl-1-methylxanthine, which are bone-resorptive agents, increased cellular cAMP and caused a slowly developing refractoriness to parathormone, prostaglandin E2, and calcitonin. Calcium (5 mM), an agent which mimics the biochemical action of parathormone in the bone cells but does not affect cAMP similarly desensitized the cells to parathormone, but not to calcitonin and only slightly to prostaglandin E2. These data suggest that heterologous desensitization of bone cells to parathormone results from the initiation of resorption by any agent regardless of whether cAMP is formed during activation of the cells. According to this concept, homologous desensitization to parathormone could involve two components: a rapid phase (< 30 min) and a long term phase, possibly stemming from initiation of resorption.     

Forskolin effects on the cAMP system and steroidogenesis in the immature rat ovary

The diterpene forskolin was found to activate the adenylate cyclase system in intact tissue and membrane preparations of the immature rat ovary. The cyclic AMP (cAMP) response reached a maximal level after 5 min and no decline was observed even after 4 h of incubation. Forskolin stimulated production of both progesterone and testosterone in a pattern similar to that produced by luteinizing hormone (LH) or dibutyryl-cAMP (dbcAMP). In combination with LH, follicle-stimulating hormone (FSH) or prostaglandin E2 (PGE2), forskolin potentiated the hormone effects on adenylate cyclase activity in membrane preparations. Pretreatment with LH or PGE2 desensitized the cells to further hormone stimulation, while the forskolin response was unaffected. Pre-exposure to forskolin did not desensitize the cells to a subsequent stimulation by LH or PGE2. The presence of 8-bromo-cAMP (brcAMP) in the preincubation medium reduced the subsequent hormone response. These results demonstrate a rapid and sustained activation of the adenylate cyclase system by forskolin in the rat ovary. The steroidogenic response was similar to that of known stimulators of ovarian cells (LH, dbcAMP). The inability of forskolin to induce desensitization of the adenylate cyclase system demonstrates, however, important differences between hormone and non-hormone activation. Consequently, forskolin can be a useful tool for investigation of the mechanisms involved in the desensitization process.     

Involvement of phosphodiesterase in the refractoriness of the Sertoli cell

Gonadotropin treatment of the Sertoli cell produces a marked refractory state of the cell to subsequent hormonal stimulation. Because FSH also stimulates the phosphodiesterase activity of these cells, the possible involvement of an altered cAMP catabolism during refractoriness was investigated in an in vitro model. Sertoli cells, after 3 days of culture in a defined medium, were exposed to FSH or isoproterenol for 1-24 h. After this pretreatment, cells were stimulated for 1 h with a maximal FSH dose, and the responsiveness was measured in terms of cAMP accumulation. Sertoli cells previously treated with hormone entered a refractory state, a second exposure being ineffective in elevating intracellular or extracellular cAMP. Addition of the phosphodiesterase inhibitor 3-isobutyl-methylxanthine in the second incubation partially restored the ability of the cell to accumulate cAMP in the presence of hormone. This phosphodiesterase inhibitor also caused an apparent decrease in the potency of FSH to induce the refractory state. Such an impairment of response developed in the intact cell in 4 h, and was accompanied by a partial desensitization of the adenylate cyclase and an increase in phosphodiesterase activity. The stimulation of phosphodiesterase activity, but not the desensitization of adenylate cyclase, was inhibited by cycloheximide. The inhibition of protein synthesis also prevented the onset of the refractory state of the intact Sertoli cell. Pretreatment of the Sertoli cells with either FSH or isoproterenol rendered the cell refractory to a second stimulation with either agonist; in contrast, the adenylate cyclase desensitization in the homogenate was apparent only for the agonist employed in the preincubation. These results indicate that phosphodiesterase regulation is involved in the control of Sertoli cell responsiveness to hormone. Thus, the net decrease in cAMP production of the FSH-treated cells is the result of a decreased adenylate cyclase stimulation and an increased cAMP catabolism mediated by phosphodiesterase. The latter phenomenon appears to be the predominant cause of the partial refractoriness induced by low doses of gonadotropin.     

Interactions between intracellular signals involved in the regulation of 25-hydroxyvitamin D3 metabolism

The tumor-promoting phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) increases 25-hydroxyvitamin D3 (25OHD3)-24-hydroxylase and decreases 25OHD3-1-hydroxylase activity in cultured kidney cells, effects similar to those exerted by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and opposite those of PTH, forskolin, and cAMP. In this paper it is shown that the effects of TPA and 1,25-(OH)2D3 are additive, suggesting that they operate through distinct mechanisms. TPA did not alter cAMP metabolism by cultured chick kidney cells, not did it alter their response, in terms of 25OHD3 metabolism, to cAMP, suggesting that these two regulators of 25OHD3 metabolism also operate through distinct pathways. Another presumed activator of protein kinase-C 1,oleoyl-2-acetyl-glycerol, was tested and found to have the same effect as TPA in decreasing 1-hydroxylase activity, but it does not increase 24-hydroxylase activity. In addition, 1-oleoyl-2-acetyl-glycerol increases intracellular cAMP levels to approximately 25% of those attained by stimulation with PTH. None of the treatments resulted in altered [3H]PDBu binding by the cells. The results, taken together, suggest that 25OHD3-1-hydroxylase and the 25OHD3-24-hydroxylase are subject to multifactorial regulation and can be regulated independently of one another.     

Resistance to parathyroid hormone-induced inhibition of inorganic phosphate transport in opossum kidney cells cultured in low inorganic phosphate medium.

Renal resistance to the phosphaturic action of parathyroid hormone (PTH) is observed during dietary deprivation of inorganic phosphate (Pi) in vivo. In the present work, the influence of short (3 h)- or long (72 h)-term deprivation of Pi on the effect of bovine PTH (bPTH(1-34)) on both Na-dependent Pi transport and cyclic AMP(cAMP) production was examined in cultured opossum kidney epithelium. Na-dependent Pi transport increased by 100% in cells exposed to low Pi medium containing no Pi (LPM) for 3 h, as compared with transport in high Pi medium containing 2 mmolPi/l (HPM). In response to a submaximal dose (1 nmol/l) of bPTH(1-34), Na-dependent Pi transport was similarly inhibited by about 40% in LPM and HPM. This inhibition was preceded by increased cAMP production which was identical in LPM and HPM. In opossum kidney cells exposed for 72 h to LPM, Na-dependent Pi transport was also increased by 100% compared with that in HPM. However, bPTH(1-34) added at 1 nmol/l did not induce any significant change in Na-dependent Pi transport or cAMP. Stimulation of cAMP could only be elicited at bPTH(1-34) concentrations higher than 1 nmol/l. Such a reduced cAMP response was also observed with forskolin in cells incubated for 72 h in LPM. The cellular resistance to the generation of cAMP was associated with a significantly lower level of ATP in cells cultured for 72 h in LPM compared with ATP levels in HPM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Forskolin increases cellular cyclic adenosine monophosphate content and parathyroid hormone release in dispersed bovine parathyroid cells

We studied the effects of the positive-inotropic and hypotensive agent forskolin on parathyroid hormone (PTH) release, cAMP accumulation, and adenylate cyclase activity in dispersed bovine parathyroid cells. Forskolin stimulated PTH release 1.7 to 3.7-fold at 10(-5) mol/L and increased cAMP content maximally 37.6-fold at 10(-4) mol/L. Forskolin induced a maximal increase in cAMP accumulation by 5 minutes of incubation. The rate of PTH release in forskolin-treated cells also reached maximal levels at five to ten minutes of incubation. There was a dose-dependent increase in PTH release and cAMP content over the range of 10(-7) to 10(-4) M forskolin with half-maximal stimulation at 10(-7) to 10(-6) mol/L for PTH release and between 10(-5) and 10(-4) mol/L for cAMP content. PTH release and cAMP accumulation were inhibited by increasing extracellular calcium concentrations in both control cells and those incubated with 10(-5) mol/L forskolin. Forskolin stimulated adenylate cyclase activity in lysates from parathyroid cells 15.7-fold with half-maximal activation between 10(-6) and 10(-5) mol/L forskolin. Forskolin-stimulated (10(-5) mol/L) and basal adenylate cyclase activities declined in parallel with increasing calcium concentrations. In contrast, phosphodiesterase activity was unaffected by forskolin at 10(-6) to 10(-4) mol/L. Forskolin, therefore, by directly activating adenylate cyclase, enhances cAMP accumulation and PTH release in dispersed bovine parathyroid cells. As with other agents activating cAMP accumulation in this system, these effects are inhibited by elevated calcium concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of phosphodiesterase inhibitors on Sertoli cell refractoriness: reversal of the impaired androgen aromatization

Responsiveness of the Sertoli cell after FSH pretreatment was evaluated in terms of androgen aromatization. Sertoli cell cultures were preincubated with FSH for 24 h, then cells were washed free of hormone and reincubated with FSH in the presence of androstendione. The estrogen accumulated in the medium was measured by RIA. Gonadotropin pretreatment produced a marked refractory state, and a second challenge with FSH did not produce an increase in androgen aromatization. A dose-response study showed that FSH pretreatment produced three separate effects on Sertoli cell steroidogenesis: an increased basal production of estrogen; a decreased maximal response when doses of 10 ng/ml FSH or higher were employed in the preincubation; and a decreased sensitivity of the Sertoli cell to FSH. In the last case, the ED50 was reduced approximately 3- to 5-fold. Such an impaired stimulation of androgen aromatization was no longer present when cells were incubated with the phosphodiesterase inhibitors methyl-isobutyl-xanthine (MIX). In the presence of this inhibitor, refractory cells responded to FSH better than the control cells. The possibility that MIX stimulated cAMP accumulation by acting as antagonist of purine receptor was ruled out by the finding that the nonxanthine phosphodiesterase inhibitor 4-(3-butoxy-4-methoxybenzyl)2-imidazolidinone (Ro 20-1724) also reverted the refractory state. Pretreatment of the Sertoli cells with FSH produced an impaired response in the second incubation also to isoproterenol, cholera toxin, and forskolin. The response to these compounds was apparently normal when cells were incubated in the presence of MIX or Ro 20-1724. Conversely, refractory cells responded to (Bu)2cAMP in a manner indistinguishable from the fully responsive control cells. These data demonstrate that FSH induces homologous and heterologous refractory states of the Sertoli cell reflected by an impaired estrogen production. The finding that phosphodiesterase inhibitors fully restore the FSH response suggests an important role of phosphodiesterase in the induction and/or maintenance of such refractoriness.     

Regulation of sodium-dependent phosphate transport by parathyroid hormone in opossum kidney cells: adenosine 3',5'-monophosphate-dependent and -independent mechanisms

The hormonal regulation of Na+-dependent phosphate transport was studied in opossum kidney (OK) cells. PTH caused time- and concentration-dependent decreases in Na+-dependent phosphate transport, with 10 pM PTH-(1-34) producing a 19% decline in phosphate transport. The EC50 for PTH inhibition of phosphate transport was 50 pM. Kinetic analyses of phosphate transport indicated that PTH decreased the maximum velocity without affecting the Km for phosphate. PTH increased cAMP formation with an EC50 of 10 nM. 8-Bromo-cAMP and (Bu)2cAMP also inhibited phosphate transport. Forskolin increased cAMP formation and decreased phosphate transport, whereas the cyclase-inactive forskolin analog 1,9-dideoxyforskolin also inhibited phosphate transport. The PTH analog [8,18-norleucine,34-tyrosinamide]PTH-(3-34) reduced phosphate transport at concentrations from 10 nM to 30 microM, but did not increase cAMP formation at concentrations up to 10 microM. The adenylate cyclase inhibitor 2',5'-dideoxyadenosine produced concentration-dependent decreases in PTH-stimulated cAMP formation, but did not influence PTH inhibition of Na+-dependent phosphate transport. Vasoactive intestinal polypeptide and prostaglandin E1 increased cAMP formation in OK cells, but were weak inhibitors of phosphate transport. This study suggests that cAMP may not be the only transmembrane signaling mechanism involved in the regulation of Na+-dependent phosphate transport by PTH-(1-34) in OK cells.     

Adenylyl cyclase of perifused porcine granulosa cells remains responsive to pulsatile, but not continuous stimulation with follicle-stimulating hormone

Differentiation of granulosa cells and development of ovarian follicles requires FSH for several days. The purpose of the present studies was to determine how the capacity of the adenylyl cyclase of immature porcine granulosa cells to respond to FSH depends on the dose, duration, and frequency of exposure to FSH. Cells were stimulated with various regimens of FSH and forskolin in a dynamic flow perifusion system. cAMP production during 4 h of continuous (tonic) exposure to FSH was directly related to FSH concentration (5-500 ng/ml). FSH-stimulated cAMP production declined markedly after 4 h of tonic stimulation with FSH, regardless of the FSH concentration. Preliminary experiments using a pulse interval of 3 h (perifusion medium FSH concentration, 150 ng/ml) indicated that stimulation with 15-min pulses elicited a greater cumulative cAMP response than stimulation with either 5-, 30-, or 60-min pulses. Responsiveness to FSH depended critically on the duration and frequency of stimulation and the concentration of FSH. Short pulses were more effective than long pulses in both eliciting cAMP responses of most uniform amplitude and maintaining responsiveness to a final tonic FSH stimulus. The optimal pattern of stimulation consisted of a pulse duration of 15 min, with a pulse interval of 2-3 h. A peak chamber FSH concentration of 150 ng/ml yielded the greatest cumulative cAMP production, although cells that had been perifused with FSH-free medium had the greatest response to a final tonic FSH stimulus. The attenuation of responsiveness after continuous perifusion with FSH does not appear to be due to desensitization of the cyclase itself, since 1) cells perifused with FSH continuously for 20 h still responded to forskolin (100 microM), which activates cyclase independently of the FSH receptor; and 2) cells did not become refractory to forskolin for 14 h. The transient refractoriness to FSH appears to be due to a process that alters the interaction between the FSH receptor and the guanine nucleotide regulatory component of cyclase. This refractoriness can be reversed simply by removing FSH from the perifusion medium for a critical period of time, i.e. 2-3 h.     

Signaling by N- and C-terminal sequences of parathyroid hormone-related protein in hippocampal neurons.

Parathyroid hormone-related protein (PTHrP) is synthesized in the brain, and a single type of cloned receptor for the N-terminal portion of PTHrP and PTH is present in the central nervous system. Nothing is known about the physiological actions or signaling pathways used by PTHrP in the brain. Using cultured rat hippocampal neurons, we demonstrate that N-terminal PTHrP[1-34] and PTH[1-34] signal via cAMP and cytosolic calcium transients. The cAMP response showed strong acute (< or = 6 h) homologous and heterologous desensitization after preincubation with PTHrP or PTH. In contrast, the acute calcium response did not desensitize after preincubation with PTHrP; in fact, preincubation dramatically recruited additional responsive neurons. Unexpectedly, C-terminal PTHrP[107-139], which does not bind or activate the cloned PTH/PTHrP receptor, signaled in neurons via cytosolic calcium but not cAMP. Although some neurons responded to both PTHrP[1-34] and PTHrP[107-139], others responded only to PTHrP[1-34]. We conclude that certain hippocampal neurons exhibit dual signaling in response to PTHrP[1-34] and that some neurons have a receptor for C-terminal PTHrP that signals only via cytosolic calcium.     

Blocking anti-thyrotropin receptor antibodies desensitize cultured human thyroid cells

Stimulating anti-TSH receptor antibodies (TSAb) mimic TSH in the induction of refractoriness in cultured thyroid cells; TSAb and TSH desensitize one another. We investigated whether blocking anti-TSH receptor antibodies (TBkAb) have the same desensitizing effects in cultured human thyroid cells. Prolonged exposure of cells (20 h) to TBkAb followed by antigen-antibody dissociation by an acid wash step was required to induce refractoriness to subsequent stimulation of cAMP accumulation with TSH and TSAb. Cycloheximide prevented this desensitization effect. The cAMP response to forskolin was not reduced in cells pretreated by TBkAb and was increased in cells desensitized by TSH or TSAb. The pattern of the TSH dose-response curves suggested that desensitization by TSH or TSAb involved only a postreceptor mechanism but both receptor and postreceptor phenomena in the case of TBkAb. In conclusion, like TSH or TSAb, TBkAb may induce a homologous desensitization in human thyroid cells which is not mediated by cAMP.