The effect of prostaglandin synthesis inhibition on the direct stimulation of renin release from rabbit renal cortical slices

Prostaglandins have been hypothesized to have several mechanistic functions in sympathetically mediated release of renin. The rabbit renal cortical slice system was chosen to examine the prostaglandin dependency of renin release directly stimulated by either a direct adenylate cyclase activator, forskolin, or a β-agonist, isoproterenol. In this study, we demonstrate that with forskolin (1 × 10⁻⁵M) or isoproterenol (1 × 10⁻⁶M), renin release was elevated 2–3 fold above control, and that this increase was shown to accompany a substantial increase in the tissue levels f cAMP (19.5 fold and 3.5 fold respectively). We also demonstrate that the increase in renin release produced by these compounds was not inhibited by cyclooxygenase inhibitors, indomethacin (25 uM) or eicosatetraynoic acid (30 ug/ml), nor was it inhibited by the selective prostacyclin synthesis inhibitor, U-51605 (30 ug/ml). Each of these inhibitors was demonstrated to block the synthesis of prostaglandins in the cortical slices at the concentrations used. Thus we propose that prostaglandins do not play a role in the induction of renin release resulting from elevated cyclic nucleotide levels or β-adrenergic stimulation.     

Effect of atriopeptin III on renin release in vitro

The ability of atriopeptin III (AP) to directly inhibit renal renin release has not been resolved. This issue was examined in a series of experiments performed in a system of rat renal cortical slices (dry weight 1.91 mg) in which the goal was to explore the effects of AP on renin release induced by cyclic AMP (cAMP)-coupled stimuli or by agents which are believed to decrease intracellular calcium (Cai). Concentration response relationships were initially established for all test agents. The cAMP stimuli utilized were isoproterenol (10(-5) M), forskolin (10(-5) M), and dibutyryl cAMP (3 X 10(-4) M); each of these agents produced a significant increase in renin release in the system (with isoproterenol a 59% increase, with forskolin 37%, and with dibutyryl cAMP 52%). The addition of AP (2.09 X 10(-8) M, a minimum inhibitory concentration derived from preliminary studies) significantly blunted these increases; in the case of the dibutyryl cAMP-stimulated renin release, the inhibition was partial as a significant 25% increase in renin occurred in the presence of AP. The addition of the calcium channel blocking agent diltiazem (10(-4) M) resulted in a significant increase in renin release (364 to 567 ng X mg-1, p less than .05) which was not blocked by the addition of AP. Similarly, TMB-8 (0.6 X 10(-4) M), another agent thought to lower Cai, also resulted in increased renin release (455 to 810 ng X mg-1), p less than .01) which was also unaffected by the addition of the AP. In summary, these results show that AP is capable of partially inhibiting renin release in vitro, particularly renin release coupled to cAMP action. In contrast, renin release induced by a decline in Cai appears to be unaffected by the addition of AP.     

Inhibition of cyclooxygenase-2: effects on renin secretion and expression in fetal lambs

The importance of prostaglandins in the regulation of the renin-angiotensin system during development is not known. These experiments were conducted to examine the effects of prostaglandin synthesis inhibitors on basal and isoproterenol-induced plasma renin concentration and renin gene expression in the late-gestation fetal lamb. Eighteen lamb fetuses ranging in gestational age from 129 to 138 days underwent surgical insertion of femoral arterial and venous catheters under general endotracheal anesthesia. After a period of recovery, animals underwent an infusion of isoproterenol after administration of a saline bolus (control experiments); 24-48 h later a second study was performed after administration of NS-398, a cyclooxygenase (COX)-2 inhibitor, or saline for a second control study. Administration of COX-2 inhibitor significantly reduced baseline plasma renin levels and attenuated responses in fetal renin secretion to isoproterenol infusions. Renal cortical cells from animals receiving COX-2 inhibitor had significantly lower levels of renin mRNA compared with animals receiving only saline. Renal cortical cells in culture from animals receiving only saline exhibited increased levels of renin mRNA when treated with isoproterenol, forskolin, or IBMX. Only forskolin increased renin mRNA levels in renal cortical cells in culture from animals receiving COX-2 inhibitor. We conclude that prostaglandins play a stimulatory role in the regulation of the renin-angiotensin system and are necessary for beta-adrenergic stimulation of renin secretion and gene expression in the late-gestation fetal lamb.     

Prostacyclin-independence in beta-adrenoceptor mediated renin release from dog renal cortical slices

There is some controversy regarding whether stimulation of renin release by the beta-adrenergic system is dependent on prostaglandin (PG) production. We have examined this problem in renal cortical slices of the dog and have obtained the following results: (1) Isoproterenol (4 X 10(-6) M) stimulated renin release, but had no effect on the formation of 6-keto PGF1 alpha, a stable metabolite of PGI2; (2) Indomethacin (2 X 10(-5) M) had no effect on isoproterenol stimulated renin release, but inhibited 6-keto PGF1 alpha formation; (3) Dibutyryl cyclic AMP (10(-3) M) stimulated both renin release, and 6-keto PGF1 alpha release. Indomethacin (2 X 10(-5) M) did not inhibit dibutyryl cyclic AMP-stimulated renin release, but did inhibit the production of 6 keto PGF1 alpha. These results indicate that the beta-adrenoceptor mediated renin release does not depend on the formation of PGI2, but renin release is dependent on cyclic AMP formation.     

Direct inhibitory effect of atriopeptin III on renin release in primate kidney

Patterns of in vitro renal renin release and the ability of atriopeptin to directly inhibit renin release have been examined in the rat, rabbit, and dog, but have been unstudied in the primate kidney. Accordingly, we examined renin release from superficial renal cortical slices of the squirrel monkey (Samiri sciuresus). The average age of the 5 animals was 10.2 +/- 2.5 yr at the time of study. Renin release was stimulated significantly by the beta-adrenergic agonist isoproterenol in concentrations of 10(-5) M (1.67-fold) and 10(-4) M (1.84-fold). Isoproterenol-induced renin release was inhibited by atriopeptin III (ANP, 2 X 10(-8) M) and the adenylate cyclase inhibitor dideoxadenosine (DDA, 10(-5) M). Similarly, the incubation of the superficial cortical slices with arachidonic acid (10(-3) M) resulted in a 4-fold increase in tissue renin release which was blocked by the calcium ionophore A23187 (17 X 10(-6) M) and ANP; interestingly, DDA did not block arachidonic acid-induced renin release. These results suggest that ANP exerts a direct inhibitory effect on B-adrenergic and arachidonic acid-induced renin release in the primate kidney. Further, the inhibitory action of A23187 on renin release suggests, as in other species, an integral role for intracellular calcium in the renin release process. These patterns of renin release in primate kidney are similar to those observed in the rodent kidney in vitro.     

Endothelin inhibits renin release from isolated rat glomeruli

The effect of endothelin on renin release from isolated rat glomeruli was examined. Endothelin inhibited basal renin release in a dose-dependent manner with an IC50 of 1.0 x 10(-9) M. Endothelin also inhibited renin release stimulated by isoproterenol (10(-5) M). Nifedipine (10(-5) M), a calcium channel blocker, induced an increase in renin release. Endothelin did not affect this nifedipine-induced renin release. These results suggest that endothelin inhibits renin release via a calcium entry mechanism and increases intracellular calcium.     

Dissociation of hyperreninemia and renal prostaglandin synthesis in the adrenalectomized rat

The aim of this study was to determine whether hyperreninemia in the adrenalectomized (ADX) rat is dependent on renal prostaglandin synthesis, as has been suggested for two other hyperreninemic conditions, Bartter's syndrome and chronic liver disease. Plasma renin concentration (PRC) in anesthetized, ADX rats was significantly increased (delta +480%; p less than 0.001) compared to sham-operated controls. In vivo, indomethacin (10 mg/kg i.v.) significantly reduced PRC of anesthetized, ADX rats after both 45 min (delta -34%; p less than 0.05) and 90 min (delta -47%; p less than 0.05). In vitro renin release from renal cortical slices of ADX rats was also significantly greater (delta +130%; p less than 0.05) than from sham-operated control cortical slices. Renin release from cortical slices of ADX rats given dexamethasone (10 micrograms/kg/day) for 4 days prior to sacrifice did not differ from sham-operated control values. Prostaglandin E2 (PGE2) release from cortical slices of ADX rats did not differ significantly from controls. However, PGE2 synthesis in glomeruli microdissected from ADX rats was significantly increased (delta +110%; p less than 0.001) compared to controls. PGE2 synthesis in glomeruli of dexamethasone-treated ADX rats remained significantly elevated compared to controls. Ibuprofen (10(-6) M) decreased PGE2 synthesis in cortical slices by 80%. However, prostaglandin synthesis inhibition had no effect on renin release from either ADX or control renal cortical slices. These results suggest that despite increased glomerular synthesis, prostaglandins do not directly influence renin release in the ADX rat.     

Hydrogen sulfide regulates cAMP homeostasis and renin degranulation in As4.1 and rat renin-rich kidney cells

The present study aims to investigate the regulatory effect of hydrogen sulfide (H(2)S) on cAMP homeostasis and renin degranulation in As4.1 and rat renin-rich kidney cells. It was found in the present study that NaHS at 0.1-10 μM significantly decreased cAMP production in As4.1 cells treated with isoproterenol (a β-adrenoceptor agonist), forskolin (an adenylyl cyclase activator), or 3-isobutyl-1-methylxanthine (IBMX, a phosphodiesterase inhibitor). NaHS at 10 μM suppressed adenylate cyclase activity but stimulated phosphodiesterase activity. We continued to study whether H(2)S may mediate cAMP-dependent renin degranulaion in As4.1 cells. It was found that NaHS at 0.1-10 μM significantly increased intracellular renin protein level. Moreover, NaHS reversed the declined renin content within As4.1 cells and normalized the upregulated renin activity in the culture medium of As4.1 cells treated with the above three stimuli. RT-PCR showed that cystathionine-γ-lyase is the main enzyme to produce endogenous H(2)S in As4.1 cells. Overexpression of cystathionine-γ-lyase increased endogenous H(2)S production and suppressed isoproterenol-induced renin release, suggesting that endogenous H(2)S may also inhibit renin release from As4.1 cells. We also tested whether H(2)S has a similar effect in renin-rich kidney cells. It was found that isoproterenol elevated intracellular cAMP level and extracellular renin activity but decreased renin protein level in the renin-rich kidney cells. Pretreatment with NaHS abolished these effects. In conclusion, H(2)S regulates cAMP homeostasis via inhibition of adenylate cyclase and stimulation of phosphodiesterase. Our findings suggest that H(2)S plays a critical role in regulation of renin degranulation in As4.1 and rat renin-rich kidney cells.     

Species differences in the effect of isoproterenol and pindolol on renin release in vitro.

The effects of selective beta adrenergic receptor stimulation with isoproterenol (3 X 10(-8) M) and of beta adrenergic blockade with pindolol (3 X 10(-5) M) on the renin release in vitro were investigated in incubated canine and rat kidney slices. Bioassay was used to measure the renin content of the tissue samples and incubation media; renin content in the canine incubation medium was measured also by radioimmunoassay. Isoproterenol in a concentration of 3 X 10(-8) M brought about a significant increase in the renin content of the incubation media as well as the tissue slices obtained from canine kidney, however, there was no change in these parameters under similar conditions if rat kidneys were incubated. Pindolol, on the other hand, in a concentration of 3 X 10(-5) M caused a significant decrease in the renin release from as well as in the renin content of the rat kidney slices, while canine kidney slices failed to respond to the same dose of the drug. The differences between the two species is suggested to be due to the differences in basal renin levels.     

Insulin modifies the properties of glucose transporters in rat brown adipose tissue.

The influence of cyclic AMP on cartilage degradation was investigated by using phosphodiesterase inhibitors [theophylline and 3-isobutyl-1-methylxanthine (IBMX)], forskolin (which activates the catalytic subunit of adenylate cyclase) and cyclic AMP analogues (dibutyryl and 8-bromo). Breakdown was assessed by quantification of proteoglycans released into the media of 8-day bovine nasal-septum cartilage cultures. Theophylline (1-20 mM), IBMX (0.01-2 mM) and dibutyryl cyclic AMP (0.1-2 mM) had little or no influence on the rate of proteoglycan release from unstimulated (no-endotoxin) cartilages. A small but detectable increase in breakdown was observed with 8-bromo cyclic AMP (0.5-2 mM) and forskolin (50-75 micrograms/ml). To examine potential inhibitory influences of these agents, the cyclic AMP modulators were added to cultures simultaneously treated with Salmonella typhosa endotoxin (12-25 micrograms/ml), a potent stimulator of cartilage degradation. The 3-4-fold stimulation of breakdown by endotoxin was strikingly inhibited by all three classes of cyclic AMP regulators. Optimal inhibition was found at 10-20 mM-theophylline, 1-2 mM-IBMX, 50-75 micrograms of forskolin/ml, 2 mM-dibutyryl cyclic AMP and 2 mM-8-bromo cyclic AMP. Inhibition was shown to be reversible, indicating that cartilages were viable after treatment. Sepharose CL-2B chromatography of proteoglycan products released from treated cartilages showed that the endotoxin-stimulated shift to lower average Mr was significantly prevented by cyclic AMP analogues and phosphodiesterase inhibitors. Together, these results show that agents which increase cyclic AMP inhibit both quantitative and qualitative aspects of endotoxin-mediated cartilage degradation.     

Effects of inhibitors of arachidonic acid metabolism on thromboplastin activity in human monocytes

Human isolated monocytes possess low levels of procoagulant activity, which was stimulated 10-30 fold by brief (2 hr) exposure to 10 micrograms/ml endotoxin. This activity was expressed in normal or factor XII-deficient plasma, but lost in plasma deficient in factors X or VII, indicating that it was due to thromboplastin. The stimulation of monocyte thromboplastin by endotoxin was inhibited in a dose-dependent manner by two phospholipase A2 inhibitors, 4-bromophenacyl bromide and quinacrine, and by two lipoxygenase inhibitors, eicosatetraynoic acid and nordihydroguaiaretic acid. Two cyclooxygenase inhibitors, aspirin and indomethacin, prevented endotoxin-induced increases in thromboxane B2 production but had no effect on thromboplastin production. These results suggest that a component in the sequence of lipid deacylation, arachidonic acid release, and metabolism via lipoxygenase may mediate the stimulation of monocyte thromboplastin activity by endotoxin.     

The inhibitory effects of calmodulin antagonists on TSH-stimulated thyroid hormone release by mouse thyroid lobes

Calcium ions have been shown to play a mojor regulatory role in the release of various hormones from a wide variety of endocrine organs. More recently, in vitro evidence suggests that a calcium-binding protein, calmodulin, is also involved in the release of many hormones. So we examined the effects of several types of calmodulin antagonists on TSH-stimulated thyroid hormone release in vitro. Mouse thyroid lobes (one thyro-tracheal unit/tube) were incubated in Krebs-Ringer bicarbonate buffer at 37 degrees C for 4h. Free thyroxine (fT4) released in the incubation medium, thyroidal cAMP and calmodulin content were measured by RIA. TSH (5 mU/ml) and dibutyryl cAMP (DBC) (200 micrograms/ml) caused a 2-4 fold increase in thyroidal release of fT4. The stimulatory effects of TSH on fT4 release were significantly inhibited by trifluoprazine and prenylamine lactate at the concentration of 5 X 10(-5) M. More specific calmodulin antagonists, W-7 and W-13, were also shown to inhibit TSH stimulation of fT4 release at the concentration of 5 X 10(-5) M. In contrast, TSH stimulation of fT4 release was not depressed by non-specific antagonists, W-5 or W-12, at the same concentration as 5 X 10(-5) M. Further, W-13 also markedly inhibited DBC-stimulated fT4 release. Neither TSH nor PGI2 altered the thyroidal calmodulin content, dissociating with a marked increase in the cAMP concentration. These results suggest that calmodulin plays an important role in TSH-stimulated thyroid hormone release and further that this mechanism exists, at least in part, at the site subsequent to the generation of cAMP.     

Bombesin enhancement of cAMP accumulation in Swiss 3T3 cells: evidence of a dual mechanism of action

Addition of bombesin in the presence of either forskolin or cholera toxin caused a marked (4-6 fold) enhancement of cAMP accumulation in Swiss 3T3 cells. This effect was time and concentration dependent, induced by various bombesin-like peptides and blocked by a bombesin antagonist. Enhancement of cAMP accumulation by bombesin was diminished by chronic pretreatment with phorbol dibutyrate implicating the involvement of protein kinase C in the activation. Pretreatment with pertussis toxin, which uncouples protein kinase C activation from cAMP accumulation (Proc. Natl. Acad. Sci. U.S.A., 84:2282, 1987) also inhibited bombesin enhancement of cAMP. Bombesin was also shown to release E type prostaglandins into the medium, an effect which was abolished by the cyclooxygenase inhibitor indomethacin. Low concentrations (100 nM) of indomethacin partially inhibited the accumulation of cAMP by bombesin in the presence of forskolin indicating that the release of E type prostaglandins into the medium is also involved in the accumulation of cAMP by bombesin. The additive nature of PBt2-mediated down-regulation and treatment with indomethacin suggests that activation of protein kinase C and the release of E type prostaglandins provide two distinct pathways involved in the enhancement of cAMP accumulation by bombesin. Finally, bombesin in the presence of forskolin stimulated the phosphorylation of the intermediate filament component vimentin, identified in the accompanying paper as a substrate for a cAMP dependent protein kinase in intact Swiss 3T3 cells.     

Prostaglandin biosynthesis does not participate in isoproterenol-induced renin release

Rat renal slices were incubated in two different media. One was a normal K, physiological saline solution and the other a high K medium. Renin release was measured every 15 min in the presence and absence of 10(-6) M isoproterenol and also in the presence and absence of aspirin, 0.8 or 1.6 X 10(-5) M. In all experiments renin release was linear during the 75 min of incubation. Isoproterenol increased renin release by approximately 100%. This was the case even in the presence of aspirin which significantly inhibited prostaglandin release (PGE2, PGF2 alpha and 6-keto-PGF1 alpha). Nor was there any reduction in the basal secretory rate by aspirin alone. These data are taken to indicate that aspirin in pharmacological doses does not interfere with either in vitro basal release rates of renin, nor the response to B agonists. It is also suggested that B agonists do not exert their effect by stimulating prostaglandin secretion.     

Forskolin and cyclic adenosine monophosphate inhibit the neurulation action of concanavalin A on explants of the gastrula ectoderm of amphibians]

We studied effects of forskolin, an activator of adenylate cyclase activity, and dioctanoyl-cyclic adenosine monophosphate (do-cAMP) on neutralizing (N) activity of concanavalin A (Con A). Biological testing was performed using explanted animal pole ectoderm of the Rana temporaria early gastrula. Con A treatment (200 micrograms/ml, 2 h) resulted in neutralization of 70-90% explants. If the explants were previously treated with forskolin (100 microM, 1 h), Con A effect decreased to 10%. When Con A and forskolin were applied simultaneously, no N-effect was observed. The same results were obtained with simultaneous treatment of the explants with Con A and do-cAMP (10(-5) M). Moreover, treatment with forskolin of the explants previously treated with Con A inhibited their neural differentiation. We suggest that N-effect of Con A is calcium dependent; the increase in intracellular cAMP after treatment of explants with forskolin or do-cAMP interferes with intracellular Ca2+ release and this results in the inhibited N-effect of Con A.     

Inhibitors of phospholipase A2 block the stimulation of protein synthesis by insulin in L6 myoblasts.

Insulin at a concentration close to the physiological range (100 mu-units/ml) stimulated protein synthesis in L6 myoblasts by 17%. Pre-treatment with the phospholipase A2 inhibitors mepacrine or dexamethasone prevented this stimulation and decreased the release of prostaglandin F2 alpha, implicating the action of phospholipase A2 and the subsequent metabolism of arachidonic acid to prostaglandins in the stimulation of protein synthesis by physiological doses of insulin. Higher concentrations of insulin (500-1000 mu-units/ml) stimulated protein synthesis in the presence of mepacrine or dexamethasone, suggesting that an alternative pathway may become important in insulin action when phospholipase A2 is inhibited.     

Developmental alterations in guanine nucleotide regulation of the beta-adrenergic receptor-adenylate cyclase system of skeletal muscle

The postnatal development of skeletal muscle is accompanied by an increased capacity for glycogenolysis and anaerobic glycolysis. In the present study, regulatory features of cAMP synthesis were examined in neonatal and adult rabbit sarcolemmal membranes. Adult sarcolemma exhibited a 3-, 6-, and 10-fold greater adenylate cyclase activity than neonate for basal, NaF, and isoproterenol plus GTP, respectively. The Km for activation by isoproterenol was 1.4 X 10(-8) M and 6 X 10(-8) M for GTP. The number of beta-receptors was similar (0.9-1.2 pmol/mg). 10 microM GTP shifted isoproterenol EC50 from 1 X 10(-8) M to 1 X 10(-7) M in adult; neonatal agonist affinity was unaffected by GTP. Cholera toxin stimulated adenylate cyclase activity 2-fold and catalyzed 32P ribosylation of a Mr = 42,000 peptide in adult sarcolemma; both activities were low or absent in neonate. Isoproterenol-stimulated GTPase activity was elevated 4-fold in adult compared to neonatal sarcolemma. Mn2+ ion-stimulated basal activity, an indicator of catalytic function of adenylate cyclase, was also elevated in adult. Together, these findings suggest that the development of catecholamine-sensitive cAMP synthesis in muscle is governed by the coordinate expression of the regulatory and catalytic proteins of adenylate cyclase, but not the beta-receptor.     

The role of beta receptors in the regulation of renin release.

The effect of the primarily beta 2 type adrenergic receptor stimulating terbutaline (10(-7)--10(-5) M) and of the beta 1 and beta 2 type adrenergic receptor stimulating isoproterenol (10(-7)--10(-5) M) was studied on renin release from incubated slices of renal cortex. Renin release and cAMP content of the slices were significantly higher in the presence of both terbutaline and isoproterenol. A logarithmic dose--response relationship was shown to be present between the beta mimetics and the renin concentration in the medium, and the cAMP content of tissue slices. In equal doses isoproterenol was about 1.5 times more potent than terbutaline. No change was seen in the renin content of the tissue slices. The results supports the view that beside the beta 1 type adrenergic receptors of the renal cortex--even if to a lesser extent--the beta 2 type adrenergic receptors, too, are involved in the regulation of renin release.     

In situ analysis of adenylate cyclase activity in permeabilized rat adipocytes: sensitivity to GTP, isoproterenol, and N6-(phenylisopropyl)adenosine

Adenylate cyclase activity in rat adipocyte suspensions was assayed in situ using a digitonin permeabilization technique. Recovery of activity was dependent on digitonin concentration, reaching a maximum at 20 micrograms/ml digitonin and paralleling the effect on cell permeability. Maximum adenylate cyclase activity recovered in permeabilized cells was 75% of that in comparable homogenates. Isoproterenol, a beta-adrenergic agonist, activated adenylate cyclase by 1.4, 2.2 and 4.5 fold at 10(-6), 10(-5) and 10(-3) M, respectively, despite perturbation of the plasma membrane. Exogenous GTP was not required for expression of beta-adrenergic activation, but 10(-5) M GTP maximally increased both basal and isoproterenol-dependent activity. The response to 10(-6) M isoproterenol was increased 2.1 fold by 10(-5) M GTP. N6-(Phenylisopropyl)adenosine at 10(-6) M inhibited both basal and isoproterenol-dependent adenylate cyclase activity by approximately 30%, demonstrating that the adenosine-dependent inhibitory pathway (Ni) remained functional in the digitonin-permeabilized cells. In situ analysis of adenylate cyclase is not only simple and rapid, but provides a unique approach to studying regulation of this key enzyme.     

Effect of forskolin on collagen production in clonal osteoblastic MC3T3-E1 cells

The effect of forskolin on collagen production in osteoblasts was investigated by using clonal osteoblastic MC3T3-E1 cells cultured in a-minimum essential medium containing 0.1% bovine serum albumin. Forskolin increased the adenylate cyclase activity in membranes pelleted from homogenates of the cell line in a dose-dependent manner. The drug caused a 13-fold stimulation at 10(-4) M, indicating that the compound directly acts on adenylate cyclase, leading to an increase in the intracellular cAMP content of the cells. Collagen accumulation in the cultures was elevated by one-day treatment with 5 X 10(-5) M forskolin to about twice that in the controls. The stimulation was mainly due to an elevation in collagen synthesis but not to an inhibition of intracellular collagen degradation because forskolin dose-dependently increased collagen synthesis; it also significantly increased the amount of low-molecular-weight hydroxyproline found in the cultures. Cells treated with forskolin produced mainly type I collagen, as found in bone matrix in situ, with only small amounts of other types of collagen. Furthermore, forskolin time-dependently inhibited DNA synthesis in the cells, indicating that the increase in type I collagen synthesis by forskolin was not due to stimulated cell proliferation. These results suggest that cAMP is closely linked to the differentiation of osteoblasts in vitro.