Correlation between cyclic AMP levels and calcium efflux in isolated renal cortical tubules.

Isolated renal cortical tubules from male hamsters were utilized to examine the possible relationship between cyclic AMP (cAMP) and efflux of calcium. Both parathyroid hormone (PTH) and prostaglandin E1 (PGE1) produced dose-related increases in cAMP levels and calcium efflux from isolated tubules. Maximal concentrations of both hormones resulted in changes in cAMP which were 6 fold greater and changes in calcium efflux which were 2 fold greater with PGE1 than with PTH. Effects of sub-maximal amounts of either hormone on both cAMP and calcium efflux were potentiated to tubule incubations resulted in increases in tissue-associated cAMP over the same degree by inclusion of methyl-isobutylxanthine (MIX). Addition of either exogenous cAMP or dibutyryl cAMP (db-cAMP) produced dose-related increases in calcium efflux which occurred more rapidly with db-cAMP than with cAMP. Increasing amounts of cAMP added to the same concentration range resulting in increases in calcium efflux. Addition of 2', 3' cyclic AMP, 5'AMP or db-cyclic GMP had no significant effect on calcium efflux while 3', 5' cyclic CMP significantly reduced this response. The results indicate that cAMP increases efflux of calcium from renal tubules and may play a central role in hormone-dependent transport of this ion.     

Cyclic adenosine 3',5'-monophosphate-induced ovulation in the perfused rat ovary and its mediation by prostaglandins

The role and mechanism of action of cyclic adenosine 3',5'-monophosphate (cAMP) in the ovulatory process was investigated by using the in vitro-perfused rat ovary model. Ovaries of pregnant mare's serum gonadotropin (PMSG, 20 IU)-primed rats were perfused for 21 h beginning in the morning of induced proestrus. In vitro stimulation with luteinizing hormone (LH; 0.1 micrograms/ml) resulted in 2.4 +/- 0.7 ovulations per treated ovary. Ovulations could also be induced by the addition of forskolin (30 microM) or dibutyryl cAMP (dbcAMP, 1 mM) with isobutylmethylxanthine (IBMX, 0.2 mM), with 11.8 +/- 1.9 and 18.6 +/- 4.4 ovulations per treated ovary, respectively. Indomethacin (5 micrograms/ml) significantly decreased the number of ovulations in the forskolin and dbcAMP + IBMX groups. The addition of prostaglandin E2 (PGE2; 1 micrograms/ml three times during the perfusion) to the forskolin + indomethacin group reversed the inhibition of ovulation (21.6 +/- 5.4 ovulations per treated ovary). Ovarian PGE tissue levels were significantly higher 10 h after stimulation with either LH, forskolin, or dbcAMP + IBMX compared to the unstimulated control group. Ovulated oocytes in the LH and forskolin groups resumed meiosis but oocytes in the dbcAMP + IBMX groups remained immature. This study shows that an increase in ovarian cAMP, even if not induced by LH, is sufficient to cause ovulation of preovulatory rat follicles, supporting the involvement of cAMP in the normal ovulatory process of the PMSG-treated rat. Furthermore, prostaglandin involvement in cAMP-induced ovulations is demonstrated.     

Pseudohypoparathyroidism showing positive phosphaturic and negative cyclic AMP excretion response to parathyroid hormone

We report a patient with pseudohypoparathyroidism (PHP) in whom parathyroid hormone (PTH) infusion failed to produce an increase in urinary adenosine 3', 5' monophosphate (cAMP) excretion in spite of the positive urinary phosphate excretion. The dbcAMP infusion test showed almost the same increase in phosphate as in the E-H test, although high urinary cAMP excretion was detected. Furthermore, a PTH infusion test in combination with calcium antagonist (diltiazem) administration markedly increased phosphate excretion, whereas the response of urinary cAMP excretion also remained negative. After treatment with 1 alpha(OH)D3, phosphaturic response increased by at least 14.3 mg/2 h compared with that in the pretreatment period. Therefore, intra and extra cellular calcium seem to affect the phosphaturic response induced by PTH.     

The effect of PGF2 alpha on parathyroid hormone-stimulated cyclic AMP production in mouse osteoblastic cell, MC3T3E1.

The effect of prostaglandin F2 alpha (PGF2 alpha) was investigated in MC3T3E1 cells on the succeeding cAMP response to parathyroid hormone (PTH). PGF2 alpha increased the membrane-associated protein kinase C (PKC) activity, indicating the activation of this enzyme. The effect of PTH to increase cAMP production was enhanced by pretreatment with PGF2 alpha. Phorbol 12-myristate 13-acetate also enhanced cAMP production stimulated by PTH, and PKC inhibitor H7 attenuated the enhancement of PGF2 alpha. A23187 did not reproduce the PGF2 alpha effect, and this effect was not antagonized by the calmodulin antagonist W7. PGF2 alpha did not change the ED50 nor the maximally responsive dose of PTH in stimulating cAMP production. The effect of PGF2 alpha was not affected by pertussis toxin, and PGF2 alpha also enhanced cholera toxin- or forskolin-stimulated cAMP production. In accordance with the response of cAMP to PTH, the resorption of mouse limb bones stimulated submaximally by PTH was enhanced by the concomitant presence of PGF2 alpha. These results indicate that PGF2 alpha modulates cAMP response through the activation of PKC, the target of which might be the catalytic unit of adenylate cyclase. Such interaction between signal transduction systems may have significance in modulating the effect of PTH on bone, i.e., bone resorption.     

Induction of metabolic changes and down regulation of bovine parathyroid hormone-responsive adenylate cyclase are dissociable in isolated osteoclastic and osteoblastic bone cells.

Bovine parathyroid hormone (PTH), dibutyryl cAMP, and calcium each induce similar metabolic changes in isolated bone cells. PTH and calcium, but not dibutyryl cAMP, result in desensitization of osteoclastic and osteoblastic bone cells to PTH. In osteoblastic cells, calcium effects are specific for PTH receptor.adenylate cyclase complexes and responsiveness to other hormones is not reduced while in osteoclastic cells, small effects of high calcium on prostaglandin E1- and epinephrine-inducible cAMP accompany the large decreases seen in cAMP response to PTH. The membrane effects of calcium and of PTH appear to be independently regulated as PTH-induced desensitization can be initiated in the absence of calcium. In addition, calcium effects on PTH-sensitive adenylate cyclase follow a different calcium dose-response than PTH-like metabolic changes. These results suggest that the effect of calcium on the membrane is not directly related to its induction of PTH-like metabolic changes. A possible role of calcium as an in vivo regulator of bone cell sensitivity to PTH is discussed.     

Effects of cholera toxin on cyclic AMP accumulation and bone resorption in cultured mouse calvaria

We have utilized the adenylate cyclase stimulator, cholera toxin, as a tool to test the role of cyclic AMP as a mediator of the effects on bone resorption by the calcium-regulating hormones, parathyroid hormone (PTH) and calcitonin. The effects on bone resorption were studied in an organ culture system using calvarial bones from newborn mice. Cyclic AMP response was assayed in calvarial bone explants and isolated osteoblasts from neonatal mouse calvaria. Cholera toxin caused a dose-dependent cAMP response in calvarial bones, seen at and above approx. 1-3 ng/ml and calculated half-maximal stimulation (EC50) at 18 ng/ml. The stimulatory effect of cholera toxin could be potentiated by the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX, 0.2 mmol/l). Cyclic AMP accumulation in the bones was maximal after 4-6 h, and thereafter declined. However, activation of the adenylate cyclase was irreversible and the total amount (bone + medium) of cAMP produced, in the presence of IBMX (0.2 mmol/l), increased with time, for at least 48 h. In osteoblast-like cells cholera toxin (1 microgram/ml) stimulated the cellular levels of cAMP with a peak after 60-120 min, which could be potentiated with IBMX. The total cAMP accumulation indicated an irreversible response. In short-term bone organ cultures (at most, 24 h) cholera toxin, at and above 3 ng/ml, inhibited the stimulatory effect of PTH (10 nmol/l) on 45Ca release from prelabelled calvarial bones. The inhibitory effect of cholera toxin (0.1 microgram/ml) on 45Ca release was significant after 6 h and the calculated IC50 value at 24 h was 11.2 ng/ml. Cholera toxin (0.1 microgram/ml) also inhibited PTH-stimulated (10 nmol/l) release of Ca2+, inorganic phosphate (Pi), beta-glucuronidase, beta-N-acetylglucosaminidase and degradation of organic matrix (release of 3H from [3H]proline-labelled bones) in 24 h cultures. 45Ca release from bones stimulated by prostaglandin E2 (1 mumol/l) and 1 alpha-hydroxyvitamin D3 (0.1 mumol/l) was also inhibited by cholera toxin (0.3 microgram/ml) in 24-h cultures. The inhibitory effect of cholera toxin on bone resorption was transient, and in long-term cultures (120 h) cholera toxin caused a dose-dependent, delayed stimulation of mineral mobilization (Ca2+, 45Ca, Pi), degradation of matrix and release of the lysosomal enzymes beta-glucuronidase and beta-N-acetylglucosaminidase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibitory effect of analogues of cyclic nucleotides and cholera toxin on relaxin release from cultured porcine luteal cells

The role of cyclic nucleotides (cyclic 3',5'-adenosine monophosphate [cAMP] and cyclic 3',5'-guanosine monophosphate [cGMP]) in the regulation of relaxin release from large porcine luteal cells was examined by use of a reverse hemolytic plaque assay. In this assay, luteal cells are cocultured in monolayers with protein-A-coupled ovine erythrocytes. In the presence of porcine relaxin antiserum and complement, a zone of hemolysis--a plaque--develops around relaxin-releasing luteal cells. The rate of development of plaques in time-course studies has been used as an index of the rate of relaxin release, and the size of plaques formed has been employed as a record of the cumulative amount of relaxin released by each cell. Treatment of monolayers with dibutyryl cAMP (dbcAMP, 60 mM) and dibutyryl GMP (dbcGMP, 15 mM resulted in a prompt inhibition in the rate of plaque formation. In addition, dbcAMP treatment reduced the average size of plaques formed. The stimulatory effect of prostaglandin F2 alpha (PGF2 alpha 10(-6) M) on relaxin release was significantly attenuated by combined treatment with dbcAMP (60 mM). Cholera toxin treatment (500 ng/ml) effectively reduced the average size of plaques formed, but neither this agent nor the beta-adrenergic agonist, isoproterenol (up to 5 X 10(-3) M), influenced the rate of plaque formation. These results--which provide evidence to show that both basal and stimulated relaxin release by large porcine luteal cells can be inhibited by the cyclic nucleotide analogues, dbcAMP and dbcGMP--are consistent with the view that these compounds have the potential to act as a negative regulatory mechanism for relaxin release.(ABSTRACT TRUNCATED AT 250 WORDS)     

The activation of cAMP-dependent protein kinase is directly linked to the stimulation of bone resorption by parathyroid hormone.

The present study was performed to characterize the direct involvement of cAMP in the stimulation of bone resorption by parathyroid hormone (PTH), using Sp-cAMPS and Rp-cAMPS, which were the direct agonist and antagonist in the activation of cAMP-dependent protein kinase (PKA), respectively. Bone resorbing activity was estimated as the number of pits formed on the dentine slice and total area of pits per slice in bone marrow cells derived from 2 week-old mice. Dibutyryl cAMP (dbcAMP)(10(-4)M) and Sp-cAMPS (10(-4)M) caused the remarkable stimulation of bone resorption. Although Rp-cAMPS (10(-4)M) did not affect bone resorption by itself, it significantly inhibited dbcAMP- and Sp-cAMPS-induced stimulation of bone resorption. Moreover, Rp-cAMPS (10(-4)M) antagonized 10(-7)M human PTH-(1-34)-induced stimulation of bone resorption, although it did not affect 10(-8)M 1,25(OH)2D3-induced stimulation of bone resorption. Present study indicates the direct involvement of PKA in the stimulation of bone resorption by PTH.     

Enhanced responsiveness to parathyroid hormone and induction of functional differentiation of cultured rabbit costal chondrocytes by a pulsed electromagnetic field

Pulsed electromagnetic fields promote healing of delayed united and ununited fractures by triggering a series of events in fibrocartilage. We examined the effects of a pulsed electromagnetic field (recurrent bursts, 15.4 Hz, of shorter pulses of an average of 2 gauss) on rabbit costal chondrocytes in culture. A pulsed electromagnetic field slightly reduced the intracellular cyclic adenosine 3',5'-monophosphate (cAMP) level in the culture. However, it significantly enhanced cAMP accumulation in response to parathyroid hormone (PTH) to 140% of that induced by PTH in its absence, while it did not affect cAMP accumulation in response to prostaglandin E1 or prostaglandin I2. The effect on cAMP accumulation in response to PTH became evident after exposure of the cultures to the pulsed electromagnetic field for 48 h, and was dependent upon the field strength. cAMP accumulation in response to PTH is followed by induction of ornithine decarboxylase, a good marker of differentiated chondrocytes, after PTH treatment for 4 h. Consistent with the enhanced cAMP accumulation, ornithine decarboxylase activity induced by PTH was also increased by the pulsed electromagnetic field to 170% of that in cells not exposed to a pulsed electromagnetic field. Furthermore, stimulation of glycosaminoglycan synthesis, a differentiated phenotype, in response to PTH was significantly enhanced by a pulsed electromagnetic field. Thus, a pulsed electromagnetic field enhanced a series of events in rabbit costal chondrocytes in response to PTH. These findings show that exposure of chondrocytes to a pulsed electromagnetic field resulted in functional differentiation of the cells.     

Adenosine 3',5'-cyclic monophosphate modulates the mitogenic responses of murine B lymphocytes

Adenosine 3',5'-cyclic monophosphate (cAMP) acts to inhibit a number of lymphocyte activities. The extent of this inhibition was tested by evaluating the effects of two cAMP-raising agents on B cell S phase entry induced by several different mitogenic regimens. It was found that both dibutyryl cAMP (dbcAMP) and isobutylmethylxanthine (IBMX) enhanced S phase entry induced by some regimens but inhibited S phase entry induced by others. The observed enhancing activity stands in contrast to the general notion of cAMP as being a "negative regulator," and it confirms that the observed inhibiting activity does not simply reflect cytotoxicity. Mitogenic regimens that appear to mimic each other, such as F(ab')2 fragments of goat anti-mouse immunoglobulin and the combination of a calcium ionophore and a phorbol ester, were distinguished by their responses to the addition of the two cAMP-raising agents. B cell responses were enhanced or inhibited even when dbcAMP was added 18-24 hr after the establishment of cultures. Cyclic AMP may regulate in a complex fashion S phase entry in cells of the immune system.     

Effect of cyclic 3':5'-AMP derivatives prostaglandins and related agents on human chorionic gonadotropin secretion in human malignant trophoblast in culture.

The secretion of human chorionic gonadotropin (hCG) is stimulated by addition of N6, O2'-dibutyryl cyclic 3':5'-AMP (dbcAMP) or theophylline to normal term placenta and human malignant trophoblast cells in vitro. To understand better the specificity of this process, malignant trophoblast cultures were incubated with 3':5'-cyclic AMP (cAMP) derivatives, prostaglandins and other agents for 1 to 3 days, and the secretion of radioimmunoassayable hCG was measured. Whereas dbcAMP was the most potent agent in stimulating secretion of hCG, the N6--and O2'-monobutyryl derivatives of cAMP and phosphodiesterase inhibitors (theophylline, papaverine, 3-isobutyl-1-methylxanthine) also increased the secretion of the hormone. A slight increase in hCG secretion was observed following addition of adenine. By contrast, butyrate, cAMP, cyclic 3':5'-GMP (cGMP), dbcGMP, 5'-AMP, adenosine, L-epinephrine and prostaglandins E1, E2, F1a and F2a were ineffective. Particulate fractions from sonicates of malignant trophoblast cultures contained adenylate cyclase activity which was stimulated more than 10-fold by NaF, but not by either catecholamines or prostaglandins. The relatively specific stimulation of hCG secretion suggested that a regulatory process involving cAMP may have physiological significance in the trophoblast.     

Effects of transforming growth factor-beta on normal clonal bone cell populations.

Although transforming growth factor-beta (TGF-beta) has been implicated in the local regulation of bone growth and remodelling, its specific effects on different subpopulations of bone cells have not been elucidated. Cells derived from bone are known to be heterogeneous and include both cells of different lineages and osteoblastic populations with different levels of expression of osteoblast-associated properties. Consequently, we have isolated clonal populations of bone cells to examine more precisely the effects of TGF-beta on individual subpopulations. Several clonal populations were isolated by limiting dilution from cells derived from 21-day-old fetal rat calvaria. Two of these clones, RCA 11 and RCB 2, were used here. While the two clones responded similarly to parathyroid hormone (PTH) and isoproterenol (ISP) with increases in intracellular cAMP, prostaglandin E2 (PGE2) elicited a 10-fold higher response in RCB 2 cells compared with RCA 11. RCB 2 cells expressed a 10-fold higher alkaline phosphatase activity compared with RCA 11. Both clones synthesized a variety of bone matrix associated proteins, but only RCA 11 synthesized SPP-1 (osteopontin) constitutively. TGF-beta stimulated growth of RCB 2 cells after 24 and 48 h of treatment, but had no effect on growth of RCA 11. TGF-beta supported anchorage-independent growth of RCB 2 cells, but not that of RCA 11. A 24-h exposure to TGF-beta decreased cAMP responsiveness to PTH and ISP slightly in both clones, but had no effect on PGE2 responses. Significant reductions in alkaline phosphatase activity were seen in both clones after 24- and 48-h treatments with TGF-beta.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of calcium administration on renal responsiveness to parathyroid hormone in pseudohypoparathyroidism type I and II -- in comparison with normals, idiopathic and surgical hypoparathyroidism.

A 31-year-old man and a 12-year-old girl were diagnosed as pseudohypoparathyroidism (PHP) Type I because of a failure to respond to the administration of parathyroid hormone (PTH) with increased urinary excretion of phosphate and cyclic adenosine-3', 5'-monophosphate (cAMP). A 22-year-old woman was diagnosed as PHP Type II because there was no increase in the urinary excretion of phosphate despite of a marked increase in urinary cAMP excretion. With the combined calcium-PTH infusion or PTH infusion after vitamin D therapy, renal response was improved in these patients. Also dibutyryl adenosine-3'-5'-cyclic monophosphate (dbcAMP) infusion evoked an increased urinary phosphate excretion in all of the patients. The metabolic defect of our patients with PHP Type I may be caused not by a lack or defective form of PTH-sensitive receptor adenylate cyclase complex but rather by an abnormal conformation in the plasma membrane-associated receptor adenylate cyclase enzyme complex in kidney. In the patient with PHP Type II, as cAMP generation is intact, the metabolic defect might be related to a defect of calcium mobilization in renal tubular cells in response to PTH.     

Interactions of Cyclic AMP and Its Dibutyryl Analogue with a Lipid Layer in the Aqueous Mixtures of Monoolein Preparation and Dioleoyl Phosphatidylcholine as Probed by X-Ray Diffraction and Raman Spectroscopy

Interactions of adenosine 3':5'-cyclicmonophosphate (cAMP) andN⁶,2'-O-dibutyryladenosine3':5'-cyclic monophosphate (dbcAMP) with alipid layer composed of monoolein-basedpreparation and dioleoylphosphatidylcholine (DOPC) wereinvestigated by small-angle X-raydiffraction (SAXD) and Raman spectroscopy.The reversed hexagonal (H_II)MO/DOPC/H₂O phase of 65:15:20 wt.%composition was selected as a referencesystem. SAXD revealed that entrapment (atthe expense of water) of 3 wt.% cAMP intothe reference system did not change thepolymorphic form and structural parametersof the phase. The same content of dbcAMPinduced the transition from the H_IIphase to the reversed bicontinuous cubicphase of space group Ia3d. Thistransition is explained by the increase oflipid head-group area due to thepenetration of the acylated adenine groupof dbcAMP into the polar/apolar region oflipid layer. The conclusion is supported byRaman spectroscopy, showing thedisruption/weakening of hydrogen bonding inthe MO/DOPC-based matrix at the N1- andN3-sites of the dbcAMP adenine ring. Asdistinct from dbcAMP, cAMP remains mostlyin the water channels of the H_IIphase, although the phosphate residue ofnucleotide interacts with the quaternaryammonium group of DOPC. Both nucleotidesincrease the population of gaucheisomers in the DOPC choline group.     

Interaction of cyclosporine A and calcitonin on bone resorption in vitro

Cyclosporine A (CsA), which is a potent immunosuppressive agent, inhibits bone resorption in vitro. The inhibition of bone resorption by CsA is sustained, unlike the transient inhibition of bone resorption produced by calcitonin (CT). These different patterns of inhibition were studied by examining the interaction between CsA and CT on stimulated bone resorption in the neonatal mouse calvarial resorptive system. "Escape" from the CT inhibition of PTH stimulated bone resorption occurred after 24 hr of organ culture. Coincubation with CsA (1 micrograms/ml) delayed the "escape" response of CT + PTH treated bones, so that the full "escape" response did not occur until after 48 hr of organ culture. Likewise, a pretreatment of 24 hr with CsA (1 micrograms/ml) was sufficient to delay "escape" from CT inhibition of PTH stimulated bone resorption until after 48 hr of organ culture. A higher concentration of CsA (10 micrograms/ml) completely prevented the "escape" response. Our data could indicate an interaction between the CsA and CT inhibitory effects on resorption.     

Interactions of cyclic AMP and its dibutyryl analogue with model membrane: X-ray diffraction and Raman spectroscopic study using cubic liquid-crystalline phases of monoolein

Interactions of adenosine 3':5'-cyclic monophosphate (cAMP) and its dibutyryl analogue, N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate (dbcAMP), with a lipid bilayer were studied by small-angle X-ray diffraction (SAXD) and Raman spectroscopy. The cubic Pn3m phase of monoolein (MO) served as a bilayer-based model system. SAXD measurements have indicated that incorporation of approximately 3 wt.% cAMP leaves the phase parameters practically unaltered, whereas the same content of dbcAMP induces the intercubic Pn3m-->Ia3d transition. By applying the concepts of lipid shape parameter and infinite periodic minimal surface to these MO phases, we have suggested that, as opposed to cAMP, dbcAMP associates with the MO bilayer. This conclusion has been supported by the different effects of phase matrix on the Raman shifts of the adenine and phosphate vibrational modes of these two nucleotides. Moreover, Raman spectra have indicated that dbcAMP inserts into the bilayer through the butyryladenine group, positioning dbcAMP preferentially at the polar/apolar interface.     

Photoaffinity labeling of parathyroid hormone receptors: comparison of receptors across species and target tissues and after desensitization to hormone

Cells derived from human giant cell tumors of bone and fibroblasts derived from human neonatal foreskin respond to parathyroid hormone (PTH) by increasing the intracellular and extracellular levels of adenosine cyclic 3',5'-phosphate (cAMP). Using photoaffinity labeling methods, we examined these cells for the presence of a PTH receptor or a binding subunit of a receptor complex. A previously designed biologically active and photolabile radioligand analogue of PTH was reacted with these intact cells. After photolysis, the cells were extracted, and the proteins were denatured, reduced, and separated by electrophoresis on sodium dodecyl sulfate (Na-DodSO4)-polyacrylamide gels followed by autoradiography. A single membrane component, Mr 70 000, was labeled specifically in intact cells cultured from skeletal and dermal tissue. By mixing, in pairs, photolabeled proteins from (a) intact human cells derived from giant cell tumors of bone, (b) intact human fibroblasts, and (c) canine renal cortical membranes, the receptors (or their binding subunits) for PTH were compared directly and found to be identical in terms of molecular size (as determined by the migration position on NaDod-SO4-polyacrylamide gels) across species (dog and human) and target tissue (bone, skin, and kidney). Preincubation of cells cultured from giant cell tumors of bone with PTH resulted in loss of the PTH-induced cAMP response (desensitization). Preincubation with PTH was accompanied by a marked decrease in photoaffinity labeling of the PTH binding component and suggests that the loss of hormone response in cells preincubated with PTH was related to a decrease in the number or availability of PTH receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrophysiological responses of osteoclasts to hormones

Electrophysiological measurements were carried out on osteoclasts in vitro. Such isolated osteoclasts are able to resorb bone in vitro and contract in response to calcitonin (CT). Our measurements show that individual osteoclasts respond to CT with a significant transient hyperpolarization of membrane potential. Application of parathyroid hormone (PTH) and dibutyryl cAMP produced a transient hyperpolarization in some osteoclasts. Measurements on an osteoblastlike line (ROS 17/2.8) showed a sustained hyperpolarizing response to CT, which is similar to but smaller than the hyperpolarizing response to PTH and dibutyryl cAMP in this and some other osteoblastlike lines. In contrast to osteoblastlike cells, the osteoclasts have no long term membrane potential response to CT, to PTH, or to dibutyryl cAMP. These results show that there are distinct differences between osteoclasts and osteoblasts in their ion transport responses to hormones.     

Effects of prostaglandin F2 alpha on agonist-induced progesterone production in cultured bovine luteal cells

The present study examines the effects of prostaglandin F2 alpha (PGF2 alpha) on basal and agonist-stimulated progesterone (P4) production utilizing long-term, serum-free cultures of bovine luteal cells. During the first 24 h of culture, PGF2 alpha had no significant effect on P4 production, and was unable to inhibit either luteinizing hormone (LH)- or dibutyryl cAMP (dbcAMP)-stimulated increases in P4. Treatment with PGF2 alpha on Day 1 produced a moderate, nonsignificant (P greater than 0.05) inhibition of cholera toxin (CT)- and forskolin (FKN)-stimulated P4 synthesis. Beyond Day 1 of culture (Days 3-11), PGF2 alpha continued to have no significant effect on basal P4 production, but suppressed all stimulatory effects of LH, dbcAMP, CT and FKN. Treatment with indomethacin inhibited prostaglandin synthesis by the cultured cells and also elevated levels of P4 from Days 3 to 11 of culture. Concurrent treatment with PGF2 alpha suppressed the steroidogenic effect of indomethacin. From these studies it was concluded that in cultured bovine luteal cells, PGF2 alpha does not affect basal P4 production, but is able to inhibit agonist-stimulated P4 production at a site beyond the accumulation of cAMP. This inhibitory effect is not apparent during the first 24 h of culture, but appears after Day 1 and persists throughout the remaining 10 days of the culture period.     

The effects of cyclic nucleotides and some related agents on 32Pi labeling of renal polyphosphoinositides in vitro.

When rabbit kidney cortex slices were incubated in the presence of ³²P_i and dibutyrylcyclic AMP (dbcAMP)⁴ a significant decrease in the labeling of phosphatidyl inositol phosphate (DPI) but not phosphatidyl inositol bisphosphate (TPI) was observed. In the presence of 0.3 mm caffeine cyclic AMP (cAMP) produced a similar effect. Caffeine potentiated the inhibitory effect of dbcAMP. At high concentrations (3 mm) caffeine alone decreased the ³²P_i labeling of both DPI and TPI. These decreases in ³²P_i labeling were not mediated by decreases in the labeling of intracellular P_i or ATP as measured by 10-min acid-labile nucleotide phosphate (10′-ALNP). Addition of cyclic GMP (cGMP) to the incubation medium decreased the labeling of DPI and to a lesser extent that of TPI also. Addition of parathyroid hormone (PTH) to the incubation medium (in the absence of exogenous cyclic nucleotides) also decreased the ³²P_i labeling of DPI but not that of TPI. In contrast to the effects of cAMP, dbcAMP, cGMP, PTH, and caffeine, the addition of insulin to the incubation medium resulted in increased ³²P_i labeling of DPI with no effect on TPI labeling. DPI isolated from kidney cortex slices prelabeled with ³²P_i and subsequently incubated with cAMP or dbcAMP contained less label than DPI isolated from slices similarly prelabeled but subsequently incubated in the absence of either cAMP or dbcAMP. These data suggest an increased rate of DPI breakdown in the presence of elevated cAMP or dbcAMP concentrations. This hypothesis was supported by the fact that cAMP stimulated the hydrolysis of DPI but not of TPI by a polyphosphoinositide phosphodiesterase present in the supernatant fraction of rabbit kidney cortex.