The hepatic clearance of circulating native and asialo carcinoembronic antigen by the rat.

Native human carcinoembryonic antigen has a circulating half life of less than 5 min when injected intravenously into rats. The intact rat accumulated carcinoembryonic antigen almost exclusively in the liver. Trace amounts were found in spleen and lung. The half life of the native glycoprotein in a rat liver perfusion system was 28 min while that of the asialo glycoprotein was 11 min. In both cases the time course for removal of glycoprotein from the perfusate was first order. After perfusion (90 min), about 10% of the glycoprotein was found in the bile. The rapid uptake of the native glycoprotein suggests that the recognition of terminal galactose may not be the only factor involved in determining the hepatic assimilation of glycoproteins.     

Cyclic nucleotide metabolism in mouse epididymal spermatozoa during capacitation in vitro

The role of cyclic nucleotides in sperm capacitation is equivocal. Using conditions known to support mouse sperm capacitation after 120 min incubation in vitro, the cAMP and cGMP contents of epididymal spermatozoa were measured and the cGMP/cAMP ratio determined. The initial high cAMP content detected upon release of spermatozoa decreased within 30 min to a lower plateau, which was then maintained throughout incubation. With the cGMP content remaining approximately constant, the cGMP/cAMP ratio increased over 120 min. In the presence of 2 mM caffeine, an increased cAMP content was noted at 0 and 30 min before a fall to the plateau level. To investigate cyclic nucleotide metabolism, adenylate cyclase and phosphodiesterase activities were compared in two sperm populations, one essentially uncapacitated and the other incubated for 120 min. Adenylate cyclase activity, higher in the presence of 2 mM Mn²⁺ compared to Mg²⁺, showed increased activity at 120 min compared to 30 min incubation, while phosphodiesterase activity decreased during this period. The ability of spermatozoa to form adenosine and inosine from cAMP indicated endogenous 5′-nucleotidase and deaminase, as well as phosphodiesterase, activities. Although the endogenous cAMP content appeared to remain constant during the time that acrosome loss, hyperactivated motility and fertilizing ability can be demonstrated, activities of the enzymes responsible for cAMP metabolism indicate an increased potential for cAMP availability and turnover. The increased cGMP/cAMP ratio may also play a role during capacitation.     

The effect of relaxin on cyclic adenosine 3',5'-monophosphate concentrations in human endometrial glandular epithelial cells

The effects of relaxin (RLX), forskolin (Fk), and 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro, a phophodeisterase inhibitor) on the accumulation of cyclic adenosine 3',5'-monophosphate (cAMP) in human endometrial glandular epithelial cells were studied. Epithelial glands were isolated from the endometrium by digesting the viable tissue fragments with collagenase. The epithelial glands were incubated with Ro, RLX, and Fk separately or in combination. The amount of cAMP was determined at the end of incubation. A moderate increase in cAMP content was observed in epithelial glands incubated with Ro alone. Accumulation of cAMP after incubation with RLX was observed only in the presence of Ro. Increase of cAMP content in response to RLX and Ro was time- and dose-dependent. The accumulation of cAMP was apparent in 5 min, reached the maximum after 15 min, and remained elevated for 17 h incubation. One nanogram per millileter RLX was effective to increase the cAMP content, with a maximal response at 100 ng/ml. The effect of Ro and the combined effect of Ro and RLX on cAMP accumulation were studied in epithelial glands of 20 endometrial specimens obtained during different stages of the menstrual cycle. When epithelial glands were incubated with Ro alone, the cAMP concentration in glands from proliferative endometria was 120 +/- 67 pmol/mg protein (n = 6, means +/- SD), significantly higher than that of secretory endometria, 42 +/- 37 (n = 14, p = 0.007). RLX and Ro caused an additional increase of cAMP accumulation, 2- to greater than 10-fold increase over the sample incubated with Ro alone. There was no significant difference between proliferative and secretory phases (500 +/- 410, n = 6, and 470 +/- 300, n = 14, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison between the effect of luteinizing hormone and prostaglandin E1 on ovarian cyclic AMP

Isolated whole ovaries from 23–24 day-old rats were studied in order to compare the effects of prostaglandin E₁ (PGE₁) and luteinizing hormone (LH) on ovarian cyclic adenosine 3′,5′-monophosphate (cAMP) production. Both substances produced a dose-dependent accumulation of cAMP in the ovarian tissue as well as in the incubation medium. The release of cAMP to the incubation medium was considerable after long periods of incubation (60–120 min). Time-relationships for LH- and PGE₁-effects were different. Maximal cAMP content in the tissue after addition of PGE₁ was seen already after 5–15 min of incubation whereas LH gave a maximal response after around 60 min. Accumulation of cAMP in the medium was approximately linear with time for both LH and PGE₁. Addition of theophylline potentiated the action of PGE₁ and LH but did not change the time-courses of the effects. It is concluded that the accumulation of cAMP in the medium should be considered in studies with various in vitro types of ovarian preparations. It is also pointed out that the different time-courses of the LH- and PGE₁-effects make the interpretation of additivity experiments difficult.     

Noradrenalin-Inducible Cyclic-AMP Accumulation in Rat Cerebral Cortex: Changes During Complete Global Ischemia

Neurologic dysfunction after cerebral ischemic insults may be due not only to neuronal death, but also to a possibly reversible failure in synaptic transmission. Because noradrenaline (NA)-inducible cyclic-AMP (cAMP) accumulation in brain may reflect the integrity of synaptic transmission mechanisms and brain viability, we studied its changes in cerebral cortex after various durations of decapitation ischemia. Unanesthetized rats were decapitated and the brains were kept at 37 degrees C for times ranging from 0 to 60 min. Cerebral cortical slices were incubated in vitro and NA (11.2 microM)-induced cAMP accumulation was evaluated over 10 min. At 0 min of ischemia, NA-induced cAMP accumulation was 56 pmol/mg protein/10 min. Between 0 and 20 min of ischemia, a linear eightfold increase, to 435 +/- 49 pmol/mg protein/10 min, occurred in NA-induced cAMP accumulation, with no further increase after longer durations of ischemia. The mechanisms modulating the increase in cortical NA-inducible cAMP accumulation with a maximum response after 20 min of ischemia remain to be defined.     

Second messenger systems in the action of LH and oxytocin on porcine endometrial cells in vitro

LH/hCG as well as oxytocin receptors are present in the porcine endometrium. Oxytocin increases phosphatidylinositol hydrolysis in this tissue, but its action on adenylate cyclase activity is disputed. The second messenger system responding to LH/hCG in endometrial cells has not been established. In this study, we investigated the involvement of protein kinase A and C signaling mechanisms in the action of LH on porcine endometrial cells in vitro. The possibility of cAMP accumulation after treatment of endometrial cells with oxytocin was also investigated. Endometrial tissue was obtained from gilts during Days 12-15 of the estrous cycle. To study the adenylate cyclase system, endometrial cells were cultured for 48 h and then incubated with different doses of LH or oxytocin for 15, 30, 60, and 180 min. To study the phospholipase C system, dispersed cells were first labeled with myo-[3H]inositol and then treated with increasing doses of LH or 100 nM of oxytocin for 30 min. Time- and dose-dependent effect of LH and oxytocin on cAMP concentration was observed. After 30 min of incubation only the highest dose of LH (100 ng/ml) was able to increase cAMP concentration in medium (P < 0.05). Longer periods (1 and 3 h) caused increased cAMP accumulation after treatment with 10 and 100 ng/ml of LH (P < 0.001). Oxytocin-stimulated cAMP concentration was observed after 1 h when only the highest dose (1000 nM) of hormone was used (P < 0.01) and after 3 h of incubation with doses of 10-1000 nM (P < 0.01). LH (10 and 100 ng/ml) increased inositol phosphates (IPs) accumulation in endometrial cells after 30 min of incubation (P < 0.01). Oxytocin involvement in IPs synthesis was more apparent than was LH (P < 0.001 versus P < 0.01). This is the first demonstration that LH receptor signaling leads to increased cAMP generation as well as IPs turnover in porcine endometrium. Oxytocin-dependent cAMP production in endometrial cells of swine was found after longer periods (3 h) of incubation. Our observations lead to the conclusion that both protein kinase A and C second messenger systems are involved in LH action and that oxytocin is able to stimulate adenylate cyclase activity in porcine endometrial cells.     

Bordetella pertussis invasive adenylate cyclase. Partial resolution and properties of its cellular penetration

The existence of an invasive adenylate cyclase in dialyzed urea extracts of the bacterium Bordetella pertussis has been suggested recently. Gel filtration of B. pertussis dialyzed urea extract shows that the invasive enzyme constitutes only a small portion of the total adenylate cyclase activity found in the extract. Its size is different than the size of the two peaks of adenylate cyclase activity identified in the extract. Ca2+ is absolutely required for the penetration of the invasive enzyme, it also controls the rate of intracellular cAMP accumulation in human lymphocytes exposed to dialyzed extract. These characteristics may be attributed to the increase in the size of the invasive enzyme as found by gel filtration chromatography of the extract in the absence of Ca2+. Removal of nonpenetrating adenylate cyclase that adheres to lymphocytes permits a direct assay of the intracellular enzyme. The time course of intracellular accumulation of adenylate cyclase activity is similar to the time course of intracellular accumulation of cAMP, suggesting that the invasive enzyme is rapidly deactivated, but not degraded, since it can be detected upon cell disruption. No appreciable amount of the enzyme is introduced when cells are incubated with extract at 4 degrees C for 120 min, then washed and incubated further at 37 degrees C. Concanavalin A inhibits cAMP accumulation irrespective of the time of its addition, and EGTA prevents penetration of the invasive enzyme even if added 20 min after addition of extract. These findings are different from those observed in other bacterial toxins thought to be internalized by receptor-mediated endocytosis. However, the cellular penetration of B. pertussis adenylate cyclase is cell-selective. It does not occur in human erythrocytes. In addition to human lymphocytes, S49 cyc- murine lymphoma, turkey erythrocytes, and rat oocytes accumulate cAMP in response to B. pertussis extract.     

Clearance of rat C-reactive protein in vivo and by perfused liver.

The clearance in vivo of rat C-reactive protein (CRP) was studied: (i) in the whole animal and (ii) by using a rat liver perfusion system. Rat CRP is a glycosylated serum protein containing a complex-type biantennary carbohydrate structure on each of its five subunits. The half-life of rat asialo CRP was approximately 5 min. More than 75% of the radioactivity associated with rat asialo CRP and asialo alpha 1-acid glycoprotein (AGP) was recovered in the liver. A small amount of radioactivity (0.8%) associated with rat CRP and rat asialo CRP was found in the lungs. Competitive inhibition of the clearance of 125I-labelled rat asialo CRP from the circulation by asialo AGP was dose dependent, and resulted in a corresponding decrease in the recovery of radioactivity associated with rat asialo CRP in the liver. This indicated that asialo AGP and rat asialo CRP were cleared by the hepatic asialoglycoprotein receptor. This observation was confirmed when the clearance of rat asialo CRP was studied using a rat liver perfusion system. Using this system, the clearance of rat asialo CRP and asialo AGP from the perfusate was inhibited by N-acetylgalactosamine, but not by phosphorylcholine, a ligand through which most of the CRP reactions are mediated. This study provides an example of a circulating serum glycoprotein containing a biantennary carbohydrate structure that is cleared by the asialoglycoprotein receptor.     

Biochemical characterization of serum thyroglobulin from patients with bone metastases from follicular carcinoma of the thyroid.

The biochemical properties of serum thyroglobulin obtained from six patients with follicular carcinoma of the thyroid and distant metastases to bone(s) have been studied. Since it is difficult to isolate sufficient thyroglobulin from serum samples, in vivo radioiodinated serum thyroglobulin obtained after radioiodine administration was used. In contrast to a sharp salting-out pattern observed with native thyroglobulin isolated from normal thyroid tissue, a broad salting-out curve was seen with metastatic serum thyroglobulin. The metastatic serum thyroglobulin eluted with low ionic strength from ion-exchange column. More than 95% of metastatic serum thyroglobulin could be bound to concanavalin-A sepharose and be eluted with 0.5 M alpha-methyl mannoside. The reactivity of metastatic serum thyroglobulin and native thyroglobulin towards concanavalin-A was comparable. Both types of thyroglobulins showed identical mobilities on sucrose linear density gradient centrifugation. The metastatic serum thyroglobulin from follicular carcinoma of the thyroid thus appears to be 19 S thyroglobulin with near normal concanavalin-A binding sugars but altered surface charges.     

Role of glycosylation on the conformation and chain dimensions of O-linked glycoproteins: light-scattering studies of ovine submaxillary mucin

The effect of carbohydrate on the conformation and chain dimensions of mucous glycoproteins was investigated by using light-scattering and circular dichroism studies of native, asialo, and deglycosylated (apo) ovine submaxillary gland mucin (OSM). OSM is a large glycoprotein that is extensively O-glycosylated by the disaccharide alpha-NeuNAc(2-6)alpha-GalNAc-O-Ser/Thr. Measurements of root mean square radius of gyration, (Rg2)1/2, and hydrodynamic radius, Rh, for OSM and its derivatives were carried out as a function of molecular weight by using static and dynamic light-scattering techniques. The results were fit to the wormlike chain model for describing the dimensions of extended polymer chains. By use of this model, values of h, the length per amino acid residue, and q, the persistence length, which is a measure of chain stiffness, were obtained. These values were then used to assess the conformation and degree of chain extension of intact OSM and its partially and totally deglycosylated derivatives. Native and asialo mucin are found to be highly extended random coils, with asialo mucin having a somewhat less extended structure than intact mucin. Upon the complete removal of the carbohydrate side chains, the extended structure characteristic of intact and asialo mucin collapses to chain dimensions typical of denatured globular proteins. Conformational analyses based on the rotational isomeric state model were also performed by using the probability maps of N-acetyl-O-(GalNAc)-Thr-N-methylamide as starting conformations for native and asialo mucin. The results suggest that both the glycosylated and nonglycosylated residues in native mucin may occupy a small region of conformational space having -90 degrees less than phi less than -60 degrees and 60 degrees less than psi less than 180 degrees, while a slightly broader range is found to fit asialo mucin. The proposed conformations obtained for these mucins are consistent with their circular dichroism spectra. Significantly larger ranges of phi and psi values were obtained for apo mucin, as would be expected from its circular dichroism spectra and increased flexibility. These results indicate the expanded mucin structure is the direct result of peptide core glycosylation. These observations together with the results of earlier studies indicate that steric interactions of the O-linked GalNAc residue with the peptide core are primarily responsible for the expanded mucin structure and that these perturbations extend to the nonglycosylated amino acid residues. This expanded mucin conformation must be a significant determinant of the viscoelastic properties of these molecules in solution.     

Effect of aging on corticosterone secretion in diestrous rats

The roles of age and prolactin (PRL) in regulating glucocorticoid secretion in diestrous rats were investigated. Adrenal zona fasciculata-reticularis (ZFR) cells from young, adult, middle (mid)-aged, and old female rats were isolated. Estrous cycle stage was determined by light microscopy after vaginal smears. Blood samples were collected from right jugular vein at 0, 30, 60, and 120 min after challenge with adrenocorticotropin (ACTH). During the diestrous phase, plasma levels of estradiol and progesterone were lower in mid-aged and old rats than in either young or adult rats. Age-dependent increases of the basal levels of plasma PRL and corticosterone were observed. No difference of ACTH-increased plasma concentrations of corticosterone was observed among young, adult, mid-aged, and old rats. Aging increased the basal, ACTH-, PRL-, forskolin (an adenylate cyclase activator)-, and 3-isobutyl-l-methylxanthine (IBMX, a non-selective phosphodiesterase inhibitor)-stimulated release of corticosterone and production of adenosine 3', 5'-cyclic monophosphate (cAMP) in ZFR cells. However, the 8-Br-cAMP (a membrane-permeable cAMP)-stimulated release of corticosterone was not affected by age. Taken together, these data indicated that aging increased corticosterone secretion in female rats during diestrous phase, which is in part due to an increase in cAMP accumulation. In conclusion, aging and PRL play a stimulatory role in the co-regulation of corticosterone secretion.     

A human melanoma-derived cell line (IGR39) with a very high number of vasoactive-intestinal-peptide (VIP) receptors. 2. Effect of VIP on cAMP production and on cell-surface VIP-binding sites

Vasoactive intestinal peptide (VIP) stimulated in a dose-dependent manner the accumulation of cAMP in human melanoma-derived cell line IGR39. The maximal effect (about 100 times the basal level) was observed with 10 nM VIP. Half-maximum cAMP production was obtained at 0.78 nM VIP. VIP-related peptides were also potent in stimulating the cAMP production in IGR39 cells. The order of potency was VIP much greater than peptide histidine-methioninamide greater than human growth-hormone-releasing factor(1-44) greater than secretin greater than glucagon. Using the same conditions, IGR37 cells, a metastasic counterpart of IGR39 cells, displayed a weak stimulation of cAMP production. After exposure of IGR39 cells to 10 nM VIP, the cAMP response to a new stimulation by VIP was strongly reduced. This desensitization of IGR39 cells to VIP was rapid (t1/2 less than 2 min) and homologous. Preincubation of IGR39 cells in the presence of native VIP induced disappearance of the VIP-binding sites at the cell surface. This phenomenon was dependent on time and VIP concentration. Maximum effect (loss of 80% of binding capacity) was obtained after exposure of the cells at 37 degrees C with a VIP concentration of 1 microM. The t1/2 of maximum disappearance was less than 2 min and the concentration of VIP giving half-maximum decrease in binding of mono[125I]iodinated VIP (125I-VIP) was 8 nM. This phenomenon was also reversible since 85% of the VIP-binding capacity could be restored in less than 1 h by incubating IGR39 cells in a VIP-free medium. The IGR39 cell line should be a useful model for further study of the structure and function of the human VIP receptor.     

Carbohydrate on human factor VIII/von Willebrand factor. Impairment of function by removal of specific galactose residues.

Human factor VIII/von Willebrand factor protein containing 120 +/- 12 nmol of sialic acid and 135 +/- 13 nmol of galactose/mg of protein was digested with neuraminidase. The affinity of native factor VIII/von Willebrand factor and its asialo form for the hepatic lectin that specifically binds asialoglycoproteins was assessed from in vitro binding experiments. Native factor VIII/von Willebrand factor exhibited negligible affinity while binding of the asialo derivative was comparable to that observed for asialo-alpha1-acid glycoprotein. Incubation of asialo-factor VIII/von Willebrand factor with Streptococcus pneumoniae beta-galactosidase removed only 62% of the galactose but abolished binding to the purified hepatic lectin. When the asialo derivative was incubated with purified beta-D-galactoside alpha2 leads to 6 sialyltransferase and CMP-[14C]NeuAc, only 61% of the galactose incorporated [14C]NeuAc. From the known specificites of these enzymes, it is concluded that galactose residues important in lectin binding are present in a terminal Gal/beta1 leads to 4GlcNAc sequence on asialo-factor VIII/von Willebrand factor. The relative ristocetin-induced platelet aggregating activity of native, asialo-, and agalacto-factor VIII/von Willebrand factor was 100:38:12, respectively, while procoagulant activity was 100:100:103.     

A potential mechanism for copper amplification of prostaglandin E2 action: attenuation of prostaglandin E2-induced efflux of cyclic AMP from median eminence explants.

It is known that prostaglandin E2 (PGE2) stimulation of LHRH release from the median eminence-arcuate nucleus (MEA) is mediated by the cAMP pathway, and a short pretreatment with copper markedly amplifies this release process. Because stimulation of cAMP accumulation is accompanied by cAMP efflux in many tissues, we considered the possibility that attenuation of cAMP efflux is one mechanism by which copper can enhance PGE2 action. When rat MEA explants were incubated in vitro, PGE2 induced a rapid (less than 2.5 min) and sustained (15 min) increase in cAMP efflux, the degree of which was a function of [PGE2]: by 5 min exposure to 10 microM PGEs2, efflux was 8-fold greater than the control (no PGE2) and it accounted for 12.4% of the total (tissue + medium) cAMP. Unlike the dramatic increase in cAMP efflux, PGE2 induced a moderate increase in cAMP content (49%) and in the incorporation of [3H] adenine into [3H] cAMP (78%); this increase was transient: it was evident after a 2.5 but not after a 5 min period of PGE2 exposure. Copper pretreatment did not alter this PGE2-induced increase in tissue cAMP content. In contrast, copper markedly inhibited (by 49%-66%) PGE2-induced cAMP efflux and this inhibition was noted regardless of the length of PGE2 exposure and PGE2 concentration. There was no evidence for hydrolysis of [3H]3',5'-cAMP included in the medium during the incubation with PGE2 with and without copper pretreatment. In summary, copper attenuated PGE2-induced cAMP efflux from MEA explants and this attenuation is not a consequence of a reduction in the availability of intracellular cAMP nor of hydrolysis of cAMP extracellularly.     

Copper-chelatin: isolation from various eucaryotic sources.

Bovine factor V was treated with highly purified neuraminidase to yield asialo factor V. Different preparations of factor V were found to exhibit heterogeneity with respect to the content of sialic acid, which ranged from 5% to 12%. The activity of asialo factor V in accelerating blood coagulation was found to be significantly greater than native factor V; thus, sialic acid is not essential for activity but may modulate its function. Asialo-factor V was 3.8 times more stable as calculated from the thermal decay constant at 37 °C. when compared to unmodified factor V.The increase in activity of factor V by thrombin was biphasic after removal of all sialic acid residues. The binding of asialo-factor V to phosphatidyl ethanolamine, as, characterized both by kinetics and by sucrose density gradient ultracentrifugation, resulted in an inactive complex. Double immunodiffusion shows that the sialic acid component of factor V is not necessary for its antigenicity.     

Stimulation of phospholipid hydrolysis and arachidonic acid mobilization in human uterine decidua cells by phorbol ester.

Vasopressin and oxytocin both stimulated inositol phosphate accumulation in isolated uterine decidua cells. Pretreatment of cells with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) prevented this agonist-induced phosphoinositide hydrolysis. TPA (0.1 microM) alone had no effect on basal inositol phosphate accumulation, but stimulated phosphoinositide deacylation, as indicated by a 2-fold increase in lysophosphatidylinositol and glycerophosphoinositol. TPA also stimulated a dose-related release of arachidonic acid from decidua-cell phospholipid [phosphatidylcholine (PC) much greater than phosphatidylinositol (PI) greater than phosphatidylethanolamine]. The phorbol ester 4 beta-phorbol 12,13-diacetate (PDA) at 0.1 microM had no effect on arachidonic acid mobilization. The TPA-stimulated increase in arachidonic acid release was apparent by 2 1/2 min (116% of control), maximal after 20 min (283% of control), and remained around this value (306% of control) after 120 min incubation. TPA also stimulated significant increases in 1,2-diacylglycerol and monoacylglycerol production at 20 and 120 min. Although the temporal increases in arachidonic acid and monoacylglycerol accumulation in the presence of TPA continued up to 120 min, that of 1,2-diacylglycerol declined after 20 min. In decidua cells prelabelled with [3H]choline, TPA also stimulated a significant decrease in radiolabelled PC after 20 min, which was accompanied by an increased release of water-soluble metabolites into the medium. Most of the radioactivity in the extracellular pool was associated with choline, whereas the main cellular water-soluble metabolite was phosphorylcholine. TPA stimulated extracellular choline accumulation to 183% and 351% of basal release after 5 and 20 min respectively and cellular phosphorylcholine production to 136% of basal values after 20 min. These results are consistent with a model in which protein kinase C activation by TPA leads to arachidonic acid mobilization from decidua-cell phospholipid by a mechanism involving phospholipase A-mediated PI hydrolysis and phospholipase C-mediated PC hydrolysis, coupled with further hydrolysis of the 1,2-diacylglycerol product.     

D1 Dopamine Receptors of NS20Y Neuroblastoma Cells Are Functionally Similar to Rat Striatal D1 Receptors

Dopamine or agonists with D1 receptor potency stimulated cyclic AMP (cAMP) accumulation in whole cell preparations of NS20Y neuroblastoma cells. The accumulation of cAMP after D1 stimulation was rapid and linear for 3 min. Both dopamine and the novel D1 receptor agonist dihydrexidine stimulated cAMP accumulation two- to three-fold over baseline. The pseudo-Km for dopamine was approximately 2 microM, whereas for dihydrexidine it was approximately 30 nM. The effects of both drugs were blocked by either the D1-selective antagonist SCH23390 (Ki, 0.3 nM) or the nonselective antagonist (+)-butaclamol (Ki, 5 nM). Both (-)-butaclamol and the D2-selective antagonist (-)-sulpiride were ineffective (Ki greater than 3 microM). Forskolin (10 microM), prostaglandin E1 (1 microM), and adenosine (10 microM) also stimulated cAMP accumulation, but none were antagonized by SCH23390 (1 microM). Finally, muscarinic receptor stimulation (100 microM carbachol) inhibited both D1- and forskolin-stimulated increases in cAMP accumulation by 80%. The present results indicate that NS20Y neuroblastoma cells have D1 receptors that are coupled to adenylate cyclase, and that these receptors have a pharmacological profile similar to that of the D1 receptor(s) found in rat striatum.     

Inhibition by vanadate of cyclic AMP production in rat corpora lutea incubated in vitro

Vanadate, a normal constituent of cells, has been reported to affect a variety of enzymes involved in phosphate transfer; the findings regarding adenylate cycle vary with the tissue and experimental system. In the corpus luteum, cyclic AMP (cAMP) stimulates steroidogenesis; and prostaglandin F2 alpha, which induces luteal regression, inhibits luteinizing hormone (LH)-induced cAMP accumulation. We examined the influence of orthovanadate on cAMP concentration in isolated corpora lutea from pseudopregnant rats. With 2 mM vanadate, basal cAMP level was unaffected, but LH-induced cAMP accumulation was inhibited by 45-68%. Lower doses of vanadate (0.2-1 mM) were almost as effective. When added simultaneously with LH, vanadate was inhibitory within 25 min, but no inhibition occurred when vanadate was added for 30 min to tissue pretreated with LH for 60 min. The decrease in cAMP accumulation was observed also when corpora lutea were exposed to vanadate in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.5 mM), indicating that vanadate inhibits cAMP synthesis. Vanadate may increase cytosolic calcium by inhibiting ion pumps in cell membranes. Thus, we examined the effect of vanadate in corpora lutea incubated in calcium-depleted medium and found that vanadate still inhibited cAMP formation. Vanadyl sulfate (0.4 and 2 mM) reduced the LH-induced cAMP accumulation as effectively as vanadate. Thus, the use of vanadate as a tool for exploring physiological regulators of luteal adenylate cyclase should be considered.     

Cross-talk regulation between cyclic AMP production and phosphoinositide hydrolysis induced by prostaglandin E2 in osteoblast-like cells.

In cloned osteoblast-like MC3T3-E1 cells, PGE2 stimulated both cAMP accumulation and the formation of inositol trisphosphate (IP3) dose dependently. The cAMP accumulation showed the peak value at 5 min and decreased thereafter, whereas the IP3 formation reached a plateau almost within 10 min and sustained it up to 30 min. The effect of PGE2 on cAMP accumulation (EC50 was 80 nM) was more potent than that on IP3 formation (EC50 was 0.8 microM). 12-O-Tetradecanoyl-phorbol-13-acetate (TPA), a protein kinase C (PKC)-activating phorbol ester, reduced the PGE2-induced cAMP accumulation, whereas 4 alpha-phorbol 12,13-didecanoate, a PKC-nonactivating phorbol ester, had little effect on the cAMP accumulation. 1-Oleoyl-2-acetyl-glycerol, a specific activator for PKC, inhibited PGE2-induced cAMP accumulation. TPA had little effect on cAMP accumulation induced by forskolin or NaF, a GTP-binding protein activator. So, the effect of TPA is presumed to be exerted at the point between the PGE2 receptor and Gs. On the other hand, forskolin and dibutyryl cAMP had little effect on the IP3 formation stimulated by PGE2. H-7, a PKC inhibitor, enhanced the PGE2-induced cAMP accumulation in comparison with HA1004, a control for H-7. Our data suggest that PGE2 regulates cAMP production through self-induced activation of PKC. These results strongly suggest that there is an autoregulatory mechanism in PGE2 signaling, and PGE2 modulates osteoblast functions through a cross-talk interaction between cAMP production and phosphoinositide hydrolysis in osteoblast-like cells.