CREB trans-activation of disruptor of telomeric silencing-1 mediates forskolin inhibition of CTGF transcription in mesangial cells.

Connective tissue growth factor (CTGF) participates in diverse fibrotic processes including glomerulosclerosis. The adenylyl cyclase agonist forskolin inhibits CTGF expression in mesangial cells by unclear mechanisms. We recently reported that the histone H3K79 methyltransferase disruptor of telomeric silencing-1 (Dot1) suppresses CTGF gene expression in collecting duct cells (J Clin Invest 117: 773-783, 2007) and HEK 293 cells (J Biol Chem In press). In the present study, we characterized the involvement of Dot1 in mediating the inhibitory effect of forskolin on CTGF transcription in mouse mesangial cells. Overexpression of Dot1 or treatment with forskolin dramatically suppressed basal CTGF mRNA levels and CTGF promoter-luciferase activity, while hypermethylating H3K79 in chromatin associated with the CTGF promoter. siRNA knockdown of Dot1 abrogated the inhibitory effect of forskolin on CTGF mRNA expression. Analysis of the Dot1 promoter sequence identified a CREB response element (CRE) at -384/-380. Overexpression of CREB enhanced forskolin-stimulated Dot1 promoter activity. A constitutively active CREB mutant (CREB-VP16) strongly induced Dot1 promoter-luciferase activity, whereas overexpression of CREBdLZ-VP16, which lacks the CREB DNA-binding domain, abolished this activation. Mutation of the -384/-380 CRE resulted in 70% lower levels of Dot1 promoter activity. ChIP assays confirmed CREB binding to the Dot1 promoter in chromatin. We conclude that forskolin stimulates CREB-mediated trans-activation of the Dot1 gene, which leads to hypermethylation of histone H3K79 at the CTGF promoter, and inhibition of CTGF transcription. These data are the first to describe regulation of the Dot1 gene, and disclose a complex network of genetic and epigenetic controls on CTGF transcription.

A pharmacological investigation of noradrenergic receptor mechanisms in neophobia

The dorsal noradrenergic bundle (DB) is a major ascending pathway which originates in the locus coeruleus of the brainstem and projects to the forebrain. The behavioral role of the DB remains unclear, despite a great deal of effort. Selective attention and anxiety are two areas which have been the focus of recent research. Some studies of the DB utilize the neurotoxin 6-hydroxydopamine (6-OHDA), since 6-OHDA injection into this pathway results in greater than 90 percent depletion of cortical and hippocampal norepinephrine (NE). Neophobia, the fear of novelty, has been reported to be either increased or decreased by 6-OHDA lesions of the DB, depending on conditions. The selective attention hypothesis would be supported by increased neophobia after 6-OHDA lesions, while the anxiety hypothesis would be supported by decreased neophobia. We have examined the effects of 6-OHDA DB lesions on neophobia under conditions in which the test environment and/or the test food were novel. We found that the lesion attenuates neophobia, defined as an increased preference for novel food, when both the environment and food were novel. The lesion had no effect on neophobia when only the environment or food was novel.^ We examined the effects of chronic intraventricular NE infusions on behavior in our neophobia test, in sham and 6-OHDA DB lesioned animals. We found that chronic NE infusions into lesioned animals significantly reversed the lesion-induced attenuation of neophobia. Sham/NE infused animals demonstrated a 40 percent greater preference for familiar food compared to sham/saline infused animals. These data suggest that infusions of NE have an effect opposite to lesion-induced attenuation of neophobia. Chronic infusions of the alpha adrenoceptor agonists had no consistent effects on neophobia. The beta adrenoceptor agonist, isoproterenol reversed the lesion-induced attenuation of neophobia but not to a statistically significant degree. Isoproterenol increased neophobia in sham animals. Forskolin, an adenylate cyclase activator, mimicked the effects of NE infusion by significantly reversing the lesion-induced attenuation of neophobia, while increasing neophobia in sham animals. These results suggest that increased release of NE during stress increases neophobia in part by stimulating beta adrenoceptors which activate adenylate cyclase. ^

cAMP-dependent protein kinase and heterologous desensitization of the adenylyl cyclase

Previous experiments had shown no differences in desensitization in cells with mutations of the adenylyl cyclase or the cAMP-dependent protein kinase and had ruled out this kinase as a mediator of desensitization; however, the assays of adenylyl cyclase had been made at high concentrations of free magnesium. The work presented in this dissertation documents a role for cAMP-dependent protein kinase which became apparent with assays at low concentrations of free magnesium. (1) The adenylyl cyclase in membranes from wild type S49 lymphoma cells showed substantial desensitization after incubation of the intact cells with low concentrations of epinephrine (5-20 nM). This desensitization was heterologous, that is it reduced the subsequent responses of the adenylyl cyclase to both epinephrine and prostaglandin-E$\sb1$. (2) The adenylyl cyclase in membranes of S49 cyc$\sp-$ cells, which do not make cAMP in response to hormones, and S49 kin$\sp-$ cells, which lack cAMP-dependent protein kinase activity, showed no heterologous desensitization following incubation of the intact cells with low concentrations of hormones. (3) Heterologous desensitization of the adenylyl cyclase was induced by incubations of wild type cells with forskolin, which activates the adenylyl cyclase downstream of the hormone receptors, or dibutyryl-cAMP, which activates the cAMP-dependent protein kinase directly. (4) Site-directed mutagenesis was used to delete the cAMP-dependent protein kinase consensus phosphorylation sequences on the $\beta$-adrenergic receptor. Heterologous desensitization occurred in intact L-cells expressing the wild type receptor or the receptor lacking the C-terminal phosphorylation site; however, only homologous desensitization occurred when the phosphorylation site on the third intracellular loop of the receptor was deleted. (5) To test directly the effects of cAMP-dependent protein kinase on the adenylyl cyclase the catalytic subunit of the kinase was purified from bovine heart and incubated with adenylyl cyclase in plasma membrane preparations. In this cell-free system the kinase caused rapid heterlogous reductions of the responsiveness of the S49 wild type adenylyl cyclase. Additionally, the adenylyl cyclase in kin$\sp-$ membranes, which showed only homologous desensitization in the intact cell, was desensitization by cell-free incubation with the kinase.^ The epinephrine responsiveness was not affected in L-cell membranes expressing the $\beta$-adrenergic receptor lacking the cAMP-dependent protein kinase consensus sequence on the third intracellular loop. ^

Regulation of adenylyl cyclase by protein kinase C and P$\sb2$ purinergic agonists

Activation of protein kinase C (PKC) causes multiple effects on adenylyl cyclase (AC), (i) an inhibition of (hormone) receptor/G$\sb{\rm s}$ coupling, consistent with PKC modification of the receptor and (ii) a postreceptor sensitization consistent with a PKC-mediated modification of the stimulatory (G$\sb{\rm s}$) or inhibitory (G$\sb{\rm i}$) G-proteins or the catalyst (C) of AC. In L cells expressing the wild-type beta-adrenergic receptor ($\beta$AR) 4-$\beta$ phorbol 12-myristate-13-acetate (PMA) caused 2-3-fold increases in the K$\sb{\rm act}$ and V$\sb{\rm max}$ for epinephrine-stimulated AC activity and an attenuation of GTP-mediated inhibition of AC. Deletion of a concensus site for PKC phosphorylation (amino acids 259-262) from the $\beta$AR eliminated the PMA-induced increase in the K$\sb{\rm act}$, but had no effect on the other actions of PMA. PMA also increased the K$\sb{\rm act}$ and V$\sb{\rm max}$ for prostaglandin E$\sb1$ (PGE$\sb1$)-stimulated AC and the V$\sb{\rm max}$ for forskolin-stimulated AC. Maximal PMA-induced sensitizations were observed when AC was assayed in the presence of 10 $\mu$M GTP and 0.3 mM (Mg$\sp{++}$).^ Liao et al. (J. Biol. Chem. 265:11273-11284 (1990)) have shown that the P$\sb2$ purinergic receptor agonist ATP stimulates hydrolysis of 4,5 inositol bisphosphate (PIP$\sb2$) by phospholipase C (PLC) in L cells. To determine if agonists that stimulate PLC and PMA had similar effects on AC function we compared the effects of ATP and PMA. ATP caused a rapid 50-150% sensitization of PGE$\sb1$-, epinephrine-, and forskolin-stimulated AC activity with an EC$\sb{50}$ of 3 $\mu$M ATP. The sensitization was similar (i.e. Mg$\sp{++}$ and GTP sensitivity) to that caused by 10 nM PMA. However, unlike PMA ATP did not affect the K$\sb{\rm act}$ for hormone-stimulated AC and its effects were unaltered by down-regulation of PKCs following long term PMA treatment. Our results demonstrate that a PKC concensus site in the $\beta$AR, is required for the PMA-induced decrease in receptor/G$\sb{\rm s}$ coupling. Our data also indicate that activation of P$\sb2$ purinergic receptors by ATP may be important in the sensitization of AC in L cells. The mechanism behind this effect remains to be determined. ^

Glutamate receptors expressed in {\it Xenopus\/} oocytes: Studies in function and regulation

The amino acid glutamate is the primary excitatory neurotransmitter for the CNS and is responsible for the majority of fast synaptic transmission. Glutamate receptors have been shown to be involved in multiple forms of synaptic plasticity such as LTP, LTD, and the formation of specific synaptic connections during development. In addition to contributing to the plasticity of the CNS, glutamate receptors also are involved in, at least in part, various pathological conditions such as epilepsy, ischemic damage due to stroke, and Huntington's chorea. The regulation of glutamate receptors, particularly the ionotropic NMDA and AMPA/KA receptors is therefore of great interest. In this body of work, glutamate receptor function and regulation by kinase activity was examined using the Xenopus oocyte which is a convenient and faithful expression system for exogenous proteins. Glutamate receptor responses were measured using the two-electrode voltage clamp technique in oocytes injected with rat total forebrain RNA. NMDA elicited currents that were glycine-dependent, subject to block by Mg$\sp{2+}$ in a voltage-dependent manner and sensitive to the specific NMDA antagonist APV in a manner consistent with those types of responses found in neural tissue. Similarly, KA-evoked currents were sensitive to the specific AMPA/KA antagonist CNQX and exhibited current voltage relationships consistent with the calcium permeable type II KA receptors found in the hippocampus. There is evidence to indicate that NMDA and AMPA/KA receptors are regulated by protein kinase A (PKA). We explored this by examining the effects of activators of PKA (forskolin, 1-isobutyl-3-methylxanthine (IBMX) and 8-Br-cAMP) on NMDA and KA currents in the oocyte. In buffer where Ca$\sp{2+}$ was replaced by 2 mM Ba$\sp{2+},$ forskolin plus IBMX and 8-Br-cAMP augmented currents due to NMDA application but not KA. This augmentation was abolished by pretreating the oocytes in the kinase inhibitor K252A. The use of chloride channel blockers resulted in attenuation of this effect indicating that Ba$\sp{2+}$ influx through the NMDA channel was activating the endogenous calcium-activated chloride current and that the cAMP mediated augmentation was at the level of the chloride channel and not the NMDA channel. This was confirmed by (1) the finding that 8-Br-cAMP increased chloride currents elicited via calcium channel activation while having no effect on the calcium channels themselves and (2) the fact that lowering the Ba$\sp{2+}$ concentration to 200 $\mu$M abolished the augmentation NMDA currents by 8-Br-cAMP. Thus PKA does not appear to modulate ionotropic glutamate receptors in our preparation. Another kinase also implicated in the regulation of NMDA receptors, calcium/phospholipid-dependent protein kinase (PKC), was examined for its effects on the NMDA receptor under low Ba$\sp{2+}$ (200 $\mu$M) conditions. Phorbol esters, activators of PKC, induced a robust potentiation of NMDA currents that was blockable by the kinase inhibitor K252A. Furthermore activation of metabotropic receptors by the selective agonist trans-ACPD, also potentiated NMDA albeit more modestly. These results indicate that neither NMDA nor KA-activated glutamate receptors are modulated by PKA in Xenopus oocytes whereas NMDA receptors appear to be augmented by PKC. Furthermore, the endogenous chloride current of the oocyte was found to be responsive to Ba$\sp{2+}$ and in addition is enhanced by PKA. Both of these latter findings are novel. In conclusion, the Xenopus oocyte is a useful expression system for the analysis of ligand-gated channel activity and the regulation of those channels by phosphorylation. ^

Receptor-mediated adenylylcyclase activation following ``homologous'' and ``heterologous'' desensitization

Using a "collision-coupling" model for $\beta \sb 2$-adrenergic receptor-mediated activation of adenylylcyclase in S49 lymphoma cells, the rate-limiting step of that activation was identified as the association of an "active-state", hormone-bound receptor (HR$\sp\*$) with a G$\sb{\rm s}$-adenylylcyclase moiety (G$\sb{\rm s}$C). It was subsequently hypothesized that the location of the rate-limiting step would not be shifted elsewhere in the activation scheme by receptor desensitization. The traditional focus of receptor desensitization studies has been on modifications of the receptor molecule itself. A "clear-cut" answer to the present hypothesis provides new information on modifications in the function of the receptor following desensitization.^ "Heterologous" desensitization was induced in wild type S49 cells with agents which increase intracellular cAMP without occupying $\beta\sb2$-adrenergic receptors; PGE$\sb1$, forskolin and dibutyryl cAMP. These treatments avoided overlapping effects on $\beta\sb2$-adrenergic receptors by the "homologous" mechanism, in which occupancy by hormone is causative. Although the steady-state activation rate was decreased following heterologous desensitization, that rate was still limited by the association between HR* and G$\sb{\rm s}$C. Thus "heterologous" desensitization acts at the equilibrium between HR and HR* (which is driven by hormone efficiency) such that HR* formation becomes less likely and the frequency of HR*G$\sb{\rm s}$C associations decreases.^ "Homologous" desensitization was induced by high (1-10$\mu$M) epinephrine concentrations in the S49 variant deficient in cAMP-dependent protein kinase, KIN$\sp-$. Use of KIN$\sp-$minimized overlapping effects by the "heterologous" mechanism, which is PKA-dependent. Following homologous desensitization, roughly 50% of the receptors in plasma membrane preparations no longer formed HR*G$\sb{\rm s}$C complexes; evidenced by a decrease in high-affinity hormone binding sites. The loss of HR*G$\sb{\rm s}$C formation did not appear related to the HR/HR* equilibrium. Increasing the efficiency of the assay agonist did nothing to "override" the effect. HR*G$\sb{\rm s}$C association was still the rate-limiting step among the remaining functional receptors. It was not distinguishable whether the remaining activity was "desensitized" due to adenylylcyclase having decreased access to receptors within plasma membrane fragments or due to an effect similar to "heterologous" desensitization. ^

THE EFFECT OF RELAXIN ON BIOCHEMICAL AND MORPHOLOGICAL PARAMETERS OF RAT MYOMETRIAL CELLS IN CULTURE (CYCLIC-AMP, CELL SHAPE CHANGE)

Relaxin is able to inhibit spontaneous, oxytocin-and prostaglandin-driven uterine contractions. The intracellular mechanism of action of relaxin on uterine relaxation had previously been studied using isometrically suspended uterine strips. Since uterine strips contain stroma as well as myometrium, the changes in biochemical parameters induced by relaxin treatment may not occur in the same cell types responsible for the physical changes. In these studies, cultures of enriched populations of rat myometrial cells were used to investigate the effect of relaxin on biochemical and morphological parameters which are related to relaxation.^ Under optimal culture conditions (initial plating density 1 - 1.5 x 10('6)cells/ml, 3 ml/35 mm dish, 2 days culture), enzymatically isolated rat myometrial cells were able to respond to relaxin with cAMP elevation. Relaxin elevated cAMP levels in the presence but not the absence of 0.1 mM methylisobutylxanthine or 0.4 um forskolin in a time- and concentration-dependent manner. In contrast, isoproterenol was able to elevate cAMP levels in the presence and absence of 0.1 mM methylisobutylxanthine.^ Oxytocin treatment caused a decrease in mean cell length and area of myometrial cells in culture which could be considered analogous to contraction. Under optimal culture conditions, relaxin increased myometrial cell length and area (i.e. analogous to relaxation) of oxytocin-treated cells in a time- and concentration-dependent manner. Other relaxants such as isoproterenol and dibutyryl cAMP also increased cell length and area of oxytocin - treated myometrial cells in culture.^ Under optimal culture conditions, relaxin decreased myosin light chain kinase activity in a time-and concentration-dependent manner by increasing the K(,50) of the enzyme for calmodulin (CaM), i.e. decreasing the affinity of the enzyme for CaM. The decrease in the affinity of myosin light chain kinase for CaM may be due to the phosphorylation of the enzyme by cAMP-dependent protein kinase. Relaxin also decreased the Ca('2+)(.)CaM-independent myosin light chain kinase activity to a lesser extent than that of the Ca('2+)(.)CaM-dependent enzyme activity. This was not attributable to a decrease in the affinity of the enzyme for myosin in myometrial cells in culture, in contrast to the finding of such a change following relaxin treatment of uterine strips. Further studies are required to clarify this point.^ There was a temporal association between the effects of relaxin on elevation of cAMP levels in the presence of 0.4 uM forskolin, increase in cell length and decrease in myosin light chain kinase activity. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI ^

cAMP regulates IL-2 receptor signaling in human T cells

Proper immune system function is dependent on positive and negative regulation of T cell signaling pathways. Full T cell activation requires sequential signaling through the T cell receptor (TCR), costimulatory molecules and the IL-2 receptor (IL-2R). The IL-2R associated Janus tyrosine kinase 3 (Jak3), as well as Signal transducer and activator of transcription 5 (Stat5), are required for normal T cell function and survival. Constitutive activation of Jak3 and Stat5 have been linked to cancers of hematopoietic origin, including certain lymphomas and leukemias. ^ The production of cAMP by adenylate cyclase has been shown to negatively regulate human TCR mediated cell proliferation. Since cAMP has been shown to negatively regulate T cell activation, we sought to investigate whether crosstalk exists between cAMP and IL-2R signaling. The first objective of this study was to determine the effect of cAMP on the activation of IL-2R signaling molecules Jak3 and Stat5. We found that the potent adenylate cyclase activator, forskolin, inhibited IL-2 activation of Jak3 and Stat5. Indeed, in vitro kinase assays and electrophoretic mobility shift assays verified a loss of Jak3 enzymatic activity and Stat5 DNA binding ability, respectively. Further analysis of IL-2R signaling showed that forskolin treatment reduced IL-2 induced association of the IL-2Rβ and γc chain. ^ Because cAMP activates protein kinase A (PKA), the second objective was to determine the role for PKA in the cAMP directed regulation of IL-2R signaling intermediates. Interestingly, forskolin induced serine phosphorylation of Jak3, suggesting that cAMP can directly regulate Jak3 via activation of a serine/threonine kinase. Indeed, phosphoamino acid analysis revealed that PKA was able to induce Jak3 serine phosphorylation in the human leukemia cell line MT-2. In addition, in vitro kinase assays established that PKA can directly inhibit Jak3 enzymatic activity. Collectively, these data indicate that cAMP negatively regulates IL-2R signaling via various effector molecules by a previously unrecognized mechanism. This new data suggests that the Jak3/Stat5 pathway may be regulated by various pharmacological agents that stimulate cAMP production and thus can be used to uncouple some types of T cell mediated diseases. ^

Regulation of v-Mos kinase activity by phosphorylation

In this thesis, I investigated the effect of cylic AMP-dependent protein kinase (PKA) on v-Mos kinase activity. Increase in PKA activity in vivo brought about either by forskolin treatment or by overexpression of the PKA catalytic subunit resulted in a significant inhibition of v-Mos kinase activity. The purified PKA catalytic subunit was able to phosphorylate recombinant p37$\rm\sp{v-mos}$ in vitro, suggesting that the mechanism of in vivo inhibition of v-Mos kinase involves direct phosphorylation by PKA. Ser-263 was identified as a residue that is normally phosphorylated at a very low level but whose phosphorylation is dramatically increased upon forskolin treatment. Consistent with the inhibitory role of Ser-263 phosphorylation, the Ala-263 mutant of v-Mos was not inhibited by forskolin treatment. Based on our results, we propose that the known inhibitory role of PKA in the initiation of oocyte maturation could be explained at least in part by its inhibition of Mos kinase.^ Combining tryptic phosphopeptide two-dimensional mapping analysis and in vitro mutagenesis studies, I identified Ser-56 as the major in vivo phosphorylation site on v-Mos. I studied the interrelationship between Ser-34 and Ser-56 phosphorylation in regulating v-Mos function. After site-directed mutagenesis to substitute serine residues with alanine or glutamic acid in different combinations to mimick unphosphorylated and phosphorylated serines respectively, various v-Mos mutants were expressed in COS-1 cells. As expected, Ala-34 mutant of v-Mos had very low (less 5% of wild type) kinase activity. The Ala-56 mutant had kinase activity 50% that of wild type. Surprisingly, the Ala-34 Ala-56 double mutant and the Ala-56 mutant exhibited identical kinase activity. On the other hand, Ala-34 Glu-56 double mutant had reduced kinase activity comparable to Ala-34 mutant. These results suggest that the phosphorylation at Ser-56 may serve to inhibit the activation of newly synthesized Mos protein. As predicted from Xenopus c-Mos studies, Glu-34 mutant of v-Mos was highly active (125% that of wild type). Interestingly, consistant with the model involving an inhibitory role of Ser-56 phosphorylation, the Glu-34 Glu-56 double mutant was totally inactive as a kinase. Moreover in my experiments, there was a perfect correlation between the level of v-Mos kinase activity of various mutants and their transforming activity. The latter is dependent upon MEK1 phosphorylation/ activation in v-mos transformed cells. Residues corresponding to both v-Mos Ser-34 and Ser-56 are evolutionarily conserved in c-Mos. Therefore, the cytostatic factor function of c-Mos may be regulated in the same manner as v-Mos kinase activity.^ It has been known that v-mos transforms cells by affecting G1 phase progression of the cell cycle. Here I showed that mos induces cyclin D1 expression in mos transformed NIH 3T3 cells and NRK 6m2 cells, and this induced level was found to be unaffected by serum starvation. Consequently, cyclin D1-Cdk4 and cyclin E-Cdk2 activities increase, and retinoblastoma protein is hyperphosphorylated. Based on studies from several laboratories, these findings suggest that increased amount of cyclin D1-Cdk4 complexes ties up the limited amount of cyclin E-Cdk2 inhibitors (e.g. p27), causing the activation of cyclin E-Cdk2. My results indicate that activation of key cell cycle regulators of G1 phase may be important for cellular transformation by mos. (Abstract shortened by UMI.) ^

Regulation of phosphatidylinositide turnover in the myometrium by cAMP: Implications for the control of uterine contraction

Regulation of uterine quiescence involves the integration of the signaling pathways regulating uterine contraction and relaxation. Uterine contractants increase intracellular calcium through receptor/GαqPLC coupling, resulting in contraction of the myometrium. Elevation of cAMP concentration has been correlated with relaxation of the myometrium. However, the mechanism of cAMP action in the uterus is unclear. ^ Both endogenous and exogenous increases in cAMP inhibited oxytocin-stimulated phosphatidylinositide turnover in an immortalized pregnant human myometrial cell line (PHM1-41). This inhibition was reversed by cAMP-dependent protein kinase (PKA) inhibitors, suggesting the involvement of PKA. cAMP inhibited phosphatidyinositide turnover stimulated by different agonists in different cell lines. These data suggest that the cAMP inhibitory mechanism is neither cell nor receptor dependent, and inhibits Gαq/PLCβ1 and PLCβ3 coupling. ^ The subcellular localization of PKA occurs via PKA binding to A-Kinase-Anchoring-Proteins (AKAP), and peptides that inhibit this association have been developed (S-Ht31). S-Ht31 blocked cAMP-stimulated PKA activity and decreased PKA concentration in PHM1-41 cell plasma membranes. S-Ht31 reversed the ability of CPT-cAMP, forskolin and relaxin to inhibit phosphatidylinositide turnover in PHM1-41 cells. Overlay analysis of both PHM1-41 cell and nonpregnant rat myometrium found an AKAPs of 86 kDa and 150 kDa associated with the plasma membrane, respectively. These data suggest that PKA anchored to the plasma membrane via AKAP150/PKA anchoring is involved in the cAMP inhibitory mechanism. ^ CPT-cAMP and isoproterenol inhibited phosphatidylinositide turnover in rat myometrium from days 12 through 20 of gestation. In contrast, neither agent was effective in the 21 day pregnant rat myometrium. The decrease in the cAMP inhibitory mechanism was correlated with a decrease in PKA and an increase in protein phosphatase 2B (PP2B) concentration in rat myometrial plasma membranes on day 21 of gestation. In myometrial total cell homogenates, both PKA and PP2B concentration increased on day 21. S-Ht31 inhibited cAMP inhibition of phosphatidylinositide turnover in day 19 pregnant rat myometrium. Both PKA and PP2B coimmunoprecipitated with an AKAP150 in a gestational dependent manner, suggesting this AKAP localizes PKA and PP2B to the plasma membrane. ^ These data presented demonstrate the importance of the cAMP inhibitory mechanism in regulating uterine contractility. ^