cAMP-activated Kinase Research Articles

Unveiling the Mechanisms of Fluid Secretion in Parotid Gland Ducts: CFTR Dependency, Cellular Contributors, and Signaling Pathways

Compelling evidence indicates that salivary gland ducts mediate fluid secretion, yet the specific cell types responsible for this process remain unclear. Our study aimed to elucidate the fundamental mechanisms governing fluid secretion within ducts and to determine their significance within the context of parotid gland function.Consistent with previous research in exocrine gland ducts, fluid secretion in parotid gland ducts is enhanced when both calcium and cAMP signaling pathways are simultaneously stimulated. Furthermore, we found that duct fluid secretion depends on the activities of CFTR, NKCC1, and the cAMP-activated kinase PKA.To further investigate the cellular contributors to duct-mediated fluid secretion, we generated a mouse model lacking ionocytes in salivary gland ducts using a diphtheria toxin-based approach. Our findings showed that ionocytes play a key role in the process of duct fluid secretion, as their absence resulted in impaired fluid secretion.Overall, our results underscore the significance of ionocytes in facilitating duct fluid secretion, highlighting the essential interplay between calcium and cAMP signaling pathways to achieve optimal secretion. Moreover, our research identified specific basolateral and apical ion-transporting proteins involved in the fluid secretion process. The pharmacological activation of ionocytes may offer a viable alternative secretory pathway when the function of secretory acinar cells is compromised, as observed in pathological conditions that lead to xerostomia. Fondecyt 1211838. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Activation of the Ae4 (Slc4a9) cation-driven Cl-/HCO3- exchanger by the cAMP-dependent protein kinase in salivary gland acinar cells.

Ae4 transporters are critical for Cl- uptake across the basolateral membrane of acinar cells in the submandibular gland (SMG). Although required for fluid secretion, little is known about the physiological regulation of Ae4. To investigate whether Ae4 is regulated by the cAMP-dependent signaling pathway, we measured Cl-/HCO3- exchanger activity in SMG acinar cells from Ae2-/- mice, which only express Ae4, and found that the Ae4-mediated activity was increased in response to β-adrenergic receptor stimulation. Moreover, pretreatment with H89, an inhibitor of the cAMP-activated kinase (PKA), prevented the stimulation of Ae4 exchangers. We then expressed Ae4 in CHO-K1 cells and found that the Ae4-mediated activity was increased when Ae4 is coexpressed with the catalytic subunit of PKA (PKAc), which is constitutively active. Ae4 sequence analysis showed two potential PKA phosphorylation serine residues located at the intracellular NH2-terminal domain according to a homology model of Ae4. NH2-terminal domain Ser residues were mutated to alanine (S173A and S273A, respectively), where the Cl-/HCO3- exchanger activity displayed by the mutant S173A was not activated by PKA. Conversely, S273A mutant kept the PKA dependency. Together, we conclude that Ae4 is stimulated by PKA in SMG acinar cells by a mechanism that probably depends on the phosphorylation of S173.NEW & NOTEWORTHY We found that Ae4 exchanger activity in secretory salivary gland acinar cells is increased upon β-adrenergic receptor stimulation. The activation of Ae4 was prevented by H89, a nonselective PKA inhibitor. Protein sequence analysis revealed two residues (S173 and S273) that are potential targets of cAMP-dependent protein kinase (PKA). Experiments in CHO-K1 cells expressing S173A and S273A mutants showed that S173A, but not S273A, is not activated by PKA.

PKA and Epac activation mediates cAMP-induced vasorelaxation by increasing endothelial NO production

Vascular relaxation induced by 3′,5′-cyclic adenosine monophosphate (cAMP) is both endothelium-dependent and endothelium-independent, although the underlying signaling pathways are not fully understood. Aiming to uncover potential mechanisms, we performed contraction–relaxation experiments on endothelium-denuded and intact rat aorta rings and measured NO levels in isolated human endothelial cells using single cell fluorescence imaging. The vasorelaxant effect of forskolin, an adenylyl cyclase activator, was decreased after selective inhibitor of protein kinase A (PKA), a cAMP-activated kinase, or L-NAME, an endothelial nitric oxide synthase (eNOS) inhibitor, only in intact aortic rings. Both selective activation of PKA with 6-Bnz-cAMP and exchange protein directly activated by cAMP (Epac) with 8-pCPT-2′–O-Me-cAMP significantly relaxed phenylephrine-induced contractions. The vasorelaxant effect of the Epac activator, but not that of the PKA activator, was reduced by endothelium removal. Forskolin, dibutyryl cAMP (a cAMP analogue), 6-Bnz-cAMP and 8-pCPT-2′–O-Me-cAMP increased NO levels in endothelial cells and the forskolin effect was significantly inhibited by inactivation of both Epac and PKA, and eNOS inhibition. Our results indicate that the endothelium-dependent component of forskolin/cAMP-induced vasorelaxation is partially mediated by an increase in endothelial NO release due to an enhanced eNOS activity through PKA and Epac activation in endothelial cells.

Abstract 3467: A novel interplay between Rap1, Epac and PKA regulates induction of angiogenesis

Abstract Advanced stage prostate cancer is associated with poor prognosis since therapeutic options are limited when the disease has progressed into a hormone refractory state. One therapeutic strategy is angiogenesis inhibition, which interferes with the blood supply of the tumor instead of targeting tumor cells directly. Here we focus on the role of three factors as regulators of angiogenesis: Rap1, a small GTPase homologous to Ras; Epac, a cAMP-activated guanine nucleotide factor for Rap1; and Protein kinase A (PKA), a cAMP-activated kinase. In previous studies, we showed that activation of Epac/Rap1 in human microvascular endothelial cells (HMVECs) inhibited VEGF-mediated chemotaxis and angiogenesis in a SCID mouse model of human angiogenesis. To test the efficacy of Epac/Rap1 activation in a prostate tumor xenograft model, we analyzed the effects of constitutive Rap1 or 8 CPT-2Me-cAMP (8CPT), a strong Epac agonist and weak PKA agonist, in PC-3 cells. PC3 cells rank high in expression levels of Rap1. Surprisingly, although 8CPT did not alter tumor growth in PC-3 cells, the drug significantly reduced tumor size in PC-3 cells expressing constitutively active Rap1. Tumor analysis showed an increase in VEGF and CD31 immunostaining in Rap1-expressing tumors, and a significant decrease in their levels upon treatment of the mice with 8CPT. To understand the mechanism of tumor inhibition, we cultured PC-3 cells under hypoxic conditions that mimic the tumor microenvironment. As observed in vivo, constitutive Rap-1 expression induced VEGF and Glut-1 mRNA levels as well as HIF-1α protein, and 8CPT reversed this effect. However, in contrast to HMVECs, 8CPT acts in PC-3 cells via PKA. A PKA activator mimicked 8CPT by inhibiting VEGF secretion in PC3-Rap1 cells under hypoxic conditions. Furthermore, the PKA inhibitor, H-89, reversed the inhibition of VEGF secretion in PC3-Rap1 cells in culture and inhibited growth in mouse xenografts. Thus, expression of Rap1 in PC-3 human prostate tumor cells promotes hypoxic induction of angiogenesis and increases the susceptibility of the cells to PKA inhibition. Taken together, our results suggest that a novel interplay between Rap1, Epac and PKA regulates tumor-microenvironment induction of angiogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3467. doi:10.1158/1538-7445.AM2011-3467

Phosphorylation of Serine 11 and Serine 92 as New Positive Regulators of Human Snail1 Function: Potential Involvement of Casein Kinase-2 and the cAMP-activated Kinase Protein Kinase A

Snail1 is a major factor for epithelial-mesenchymal transition (EMT), an important event in tumor metastasis and in other pathologies. Snail1 is tightly regulated at transcriptional and posttranscriptional levels. Control of Snail1 protein stability and nuclear export by GSK3beta phosphorylation is important for Snail1 functionality. Stabilization mechanisms independent of GSK3beta have also been reported, including interaction with LOXL2 or regulation of the COP9 signalosome by inflammatory signals. To get further insights into the role of Snail1 phosphorylation, we have performed an in-depth analysis of in vivo human Snail1 phosphorylation combined with mutational studies. We identify new phosphorylation sites at serines 11, 82, and 92 and confirmed previously suggested phosphorylations at serine 104 and 107. Serines 11 and 92 participate in the control of Snail1 stability and positively regulate Snail1 repressive function and its interaction with mSin3A corepressor. Furthermore, serines 11 and 92 are required for Snail1-mediated EMT and cell viability, respectively. PKA and CK2 have been characterized as the main kinases responsible for in vitro Snail1 phosphorylation at serine 11 and 92, respectively. These results highlight serines 11 and 92 as new players in Snail1 regulation and suggest the participation of CK2 and PKA in the modulation of Snail1 functionality.

PKA inhibits the Na +–K +pump in cadiac myocytes via superoxide

Adrenergic Na+-K+ pump regulation is often attributed to direct phosphorylation of the Na+-K+ pump molecule by cAMP-activated kinase (a.k.a. PKA) However, PKA has poor access to putative phosphorylation sites on the pump. Since reactive oxygen species (ROC) can alter function of the pump's isolated enzymatic equivalent, Na+-K+ ATPase, we examined if ROC may mediate adrenergic pump regulation in intact cells. We voltage clamped and internally perfused isolated rabbit ventricular myocytes using the whole-cell patch clamp technique.

Metabolic Pathways for Ion Homeostasis and Persistent Na+ Current

ATP supply in heart cells is preserved by a number of different mechanisms to maintain a constant level of ATP concentration. ADP, phosphocreatine, inorganic phosphate, and cAMP-activated kinases are effectively involved in ATP supply in abnormal conditions such as ischemia. Intracellular Na(+) and Ca(2+) concentrations in the heart cells are maintained at low levels through the operation of ion transport across the plasma membrane, such as Na(+)-K(+) pumps, as well as Na(+)-Ca(2+), and Na(+)-H(+) exchangers. The activity of the latter two exchanger mechanisms depends on their expression levels and the concentration gradients of Na(+) and Ca(2+) across the membrane. These exchangers may interact, and both are strongly regulated by intracellular Na(+), which is maintained by the Na(+)-K(+) pump, utilizing ATP as an energy source. The mitochondria and the sarcoplasmic reticulum are the organelles responsible for intracellular Ca(2+) stores and release sites for maintaining very low cytoplasmic concentration of Ca(2+). Ischemia disrupts the delicate interactions of these transport mechanisms. This may cause intracellular Na(+) and Ca(2+) accumulation, which can cause decreased contractility and electrical instability. Further, a persistent (noninactivating) Na(+) current may be enhanced during ischemia, and this can contribute to action potential prolongation, and the development of early afterdepolarizations. The Na(+) influx via the persistent Na(+) current may induce further Na(+) and Ca(2+) accumulation in the cells.

Formation of Inactive cAMP-saturated Holoenzyme of cAMP-dependent Protein Kinase under Physiological Conditions

The complex of the subunits (RIalpha, Calpha) of cAMP-dependent protein kinase I (cA-PKI) was much more stable (K(d) = 0.25 microm) in the presence of excess cAMP than previously thought. The ternary complex of C subunit with cAMP-saturated RIalpha or RIIalpha was devoid of catalytic activity against either peptide or physiological protein substrates. The ternary complex was destabilized by protein kinase substrate. Extrapolation from the in vitro data suggested about one-fourth of the C subunit to be in ternary complex in maximally cAMP-stimulated cells. Cells overexpressing either RIalpha or RIIalpha showed decreased CRE-dependent gene induction in response to maximal cAMP stimulation. This could be explained by enhanced ternary complex formation. Modulation of ternary complex formation by the level of R subunit may represent a novel way of regulating the cAMP kinase activity in maximally cAMP-stimulated cells.

CGMP-kinase mediates cGMP- and cAMP-induced Ca 2+ desensitization of skinned rat artery

(Rp)-8-Bromo-guanosine 3′,5′-cyclic monophosphorothioate (Rp-8-Br-cGMPS) inhibited competitively both isozymes of type Iα and Iβ cGMP-dependent protein kinase (cGMP-kinase) purified from porcine aorta with apparent K i values (μM) of 3.7 for Iα and 1.8 for Iβ. The compound also inhibited bovine heart type II cAMP-dependent protein kinase (cAMP-kinase), but with a K i of 25 μM. Thus, it is a selective inhibitor of cGMP-kinase. In α-toxin-skinned smooth muscle preparations from rat mesenteric artery, 8-Br-cGMP (10 −7 M) and 8-Br-cAMP (10 −6 M) produced a rightward shift of the concentration-contraction curves for Ca 2+, denoting a decrease in Ca 2+ sensitivity of the contractile elements. The shift by 8-Br-cAMP as well as by 8-Br-cGMP was completely reversed by Rp-8-Br-cGMPS, while a selective inhibitor of activation of cAMP-kinase, (Rp)-adenosine-3′,5′-cyclic monophosphorothioate (Rp-cAMPS), was without effects on the shift produced by these two compounds. These findings indicate the pivotal role that the activation of cGMP-kinase plays in the production of a decrease in Ca 2+ sensitivity of contractile elements. © 1997 Elsevier Science B.V. All rights reserved.

Neuropeptide-Induced Inhibition of Steroidogenesis in Crab Molting Glands: Involvement of cGMP-Dependent Protein Kinase

In crustaceans, ecdysteroid production by the molting glands (Y-organs) is negatively regulated by a neuropeptide, molt-inhibiting hormone (MIH). The involvement of cyclic nucleotide-dependent kinases in the mechanism of action of this neuropeptide was investigated with regard to the steroidogenic activity ofCarcinus maenasY-organs. Regardless of the activity level, the major phosphotransferase activity measured in cytosolic fraction was cGMP-dependent, indicating a relatively high cytosolic concentration of cGMP-kinase in these cells. Phosphotransferase activity was nearly twofold higher in the intermolt (low steroidogenic activity) than in premolt (high steroidogenic activity) animals.In vitroincubation of premolt Y-organs with MIH for 1 hr increased by 3.7-fold the cGMP-kinase activity ratio (−cGMP/+cGMP). Numerous endogenous protein substrates were predominantly phosphorylated in a cGMP-dependent manner in cytosolic, particulate, and membrane fractions. Similar phosphoprotein patterns were observed in both molting stages. By contrast, cAMP-kinase activity, which was low in intermolt Y-organs, increased significantly in the active steroidogenic premolt Y-organs. The increase in cAMP-kinase activity was accompanied by a cAMP-dependent phosphorylation of several specific endogenous proteins. Taken together these results strongly suggest that activation of cGMP-kinase and subsequent phosphorylation of an endogenous protein(s) may be responsible, at least in part, for the MIH-induced inhibition of steroidogenesis. By contrast, it is most unlikely that cAMP-kinase is involved in these processes.

Novel (Rp)-cAMPS Analogs as Tools for Inhibition of cAMP-kinase in Cell Culture: BASAL cAMP-KINASE ACTIVITY MODULATES INTERLEUKIN-1β ACTION

Novel (Rp)-cAMPS analogs differed widely in ability to antagonize cAMP activation of pure cAMP-dependent protein kinase I and II and to antagonize actions of cAMP on gene expression, shape change, apoptosis, DNA replication, and protein phosphorylation in intact cells. These differences were related to different abilities of the analogs to stabilize the holoenzyme form relative to the dissociated form of cAMP kinase type I and II. (Rp)-8-Br-cAMPS and (Rp)-8-Cl-cAMPS were the most potent cAMP antagonists for isolated type I kinase and for cells expressing mostly type I kinase, like IPC-81 leukemia cells, fibroblasts transfected with type I regulatory subunit (RI), and primary hepatocytes. It is proposed that (Rp)-8-Br-cAMPS or (Rp)-8-Cl-cAMPS should replace (Rp)-cAMPS as the first line cAMP antagonist, particularly for studies in cells expressing predominantly type I kinase. The phosphorylation of endogenous hepatocyte proteins was affected oppositely by (Rp)-8-Br-cAMPS and increased cAMP, indicating that (Rp)-8-Br-cAMPS inhibited basal cAMP-kinase activity. The inhibition of basal kinase activity was accompanied by enhanced DNA replication, an effect which could be reproduced by microinjected mutant cAMP-subresponsive RI. It is concluded that the basal cAMP-kinase activity exerts a tonic inhibition of hepatocyte replication. (Rp)-8-Br-cAMPS and microinjected RI also desensitized hepatocytes toward inhibition of DNA synthesis by interleukin-1 beta. This indicates that basal cAMP-kinase activity can have a permissive role for the action of another (interleukin-1 beta) signaling pathway.

Glycolipids isolated from Aplysia kurodai can activate cyclic adenosine 3',5'-monophosphate-dependent protein kinase from rat brain.

Cyclic AMP (cAMP)-dependent protein kinase (cAMP-kinase) partially purified from the membrane fractions of rat brains was stimulated by novel phosphonoglycosphingolipids (glycolipids) derived from the skin and nerve fibers of Aplysia kurodai. Among various glycolipids tested, a major glycolipid from the skin, 3-O-MeGal beta 1-->3GalNAc alpha 1-->3[6'-O-(2-aminoethylphosphonyl)Gal alpha 1-->2](2-aminoethylphosphonyl-->6)Glc beta 1-->4Glc beta 1-->1ceramide (SGL-II), was most potent, giving half-maximal activation at 32.2 microM. Activation of cAMP-kinase was maximal with 250 microM SGL-II using kemptide as substrate. The effect of SGL-II was additive on kinase activity at submaximal concentrations of cAMP. The kinase activity activated with SGL-II was inhibited by the addition of protein kinase inhibitor peptide, a specific peptide inhibitor for cAMP-kinase. Its inhibitory pattern was similar to that for the catalytic subunit. Of the various substrates tested, the glycolipid-stimulated cAMP-kinase could phosphorylate microtubule-associated protein 2, synapsin I, and myelin basic protein but not histone H1 and casein. The regulatory subunit strongly inhibited the activity of purified catalytic subunit of cAMP-kinase. This inhibition was reversed by addition of SGL-II, as observed for cAMP. SGL-II was capable of partially dissociating cAMP-kinase, which was observed by gel filtration column chromatography. However, the binding activity of cAMP to the holoenzyme was not inhibited with SGL-II. These results demonstrate that the glycolipids can directly activate cAMP-kinase in a manner similar, but not identical, to that of cAMP.

The regulatory diversity of the mammalian adenylyl cyclases

Clones for six mammalian adenylyl cyclases have recently been isolated. One of these enzymes, the type I calmodulin-sensitive adenylyl cyclase, is neurospecific and is implicated in neuroplasticity. We propose that the type I adenylyl cyclase may be important for learning and memory because it allows Ca 2+-amplified cAMP signals, synergism between Ca 2+ and cAMP-activated kinases, and positive feedback regulation of Ca 2+ channels by cAMP-dependent protein kinase.

TYR-MIF-1, but not MIF-1 or morphine, decreases cyclic AMP-dependent protein kinase activity extracted from HeLa cells

The biochemical effects of Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH 2), a naturally occurring opiate modulator, on cellular function are poorly understood. We looked for changes in cyclic AMP-dependent protein kinase (cAMP-kinase; protein kinase A) after treating HeLa cells with this peptide. Binding sites for Tyr-MIF-1 (IC 50 = 83 n M), but not for DAMGO, a mu opiate, were found on these cells. Brief incubation of HeLa cells with 10 −7 to 10 −5 M Tyr-MIF-1 significantly decreased, in a rapid and dose-dependent manner, cAMP-kinase activity in cell extracts assayed in reaction mixtures with added cAMP but not the much lower basal enzymatic activity that is dependent on endogenous cAMP. In contrast, incubation of the cells with the related peptide MIF-1 (Pro-Leu-Gly-NH 2) or with morphine did not significantly alter cAMP-kinase activity. This study shows that Tyr-MIF-1 can affect part of a major signal transduction pathway independently of binding to opiate receptors.

Programmed cell death (apoptosis) is induced rapidly and with positive cooperativity by activation of cyclic adenosine monophosphate‐kinase I in a myeloid leukemia cell line

Programmed death (apoptosis) of the rat myelocytic leukemic cell line IPC-81 was triggered by cyclic adenosine monophosphate (cAMP) analogs or by agents (cholera toxin, prostaglandins) increasing the endogenous cAMP level. The induction of cell death by cholera toxin was preceded by increased activation of cAMP-kinase. Cell lysis started already 5 hr after cAMP challenge and was preceded by internucleosomal DNA fragmentation and morphological changes characteristic of apoptosis. The cell suicide could be prevented by inhibitors of macromolecular synthesis. cAMP analogs induced cell death in a positively cooperative manner (apparent Hill coefficient of 2.9), indicating that triggering of the apoptotic process was under stringent control. There was a strong synergism between cAMP analogs complementing each other in the activation of cAMP-dependent protein kinase I (cAKI). No such synergism was noted for analogs complementing each other in the activation of cAKII. It is concluded that apoptosis can be induced solely by activation of cAKI. The IPC-81 cells expressed about four times more cAKI than cAKII. The expression of cAK subunits, on the protein and mRNA levels, was only minimally affected by cholera toxin treatment.

Activation of microsomal c AMP-dependent protein kinase isoenzyme I by ACTH 1–24 in bovine adrenal cells

In microsomes of bovine fasciculata reticularis cells incubated with or without 10 −8 M ACTH during 20 min, we measured covalent and non covalent cAMP binding under exchange or non-exchange conditions and cAMP-kinase activity. ACTH induced a decrease in cAMP-kinase activity and in the number of free cAMP binding sites. These results indicate an activation by ACTH of a part ofmicrosomal cAMP-dependent protein kinase. Photoaffinity labeling of microsomal protein with 8-azido- cAMP revealed the presence of both cAMP-kinase isoenzyme I and II in this cellular fraction. Using this method, it was demonstrated that ACTH 1–24 caused a preferential and nearly complete activation ofmicrosomal protein kinase I.

Activation of protein kinase activity in pancreatic acini by calcium and cAMP.

Regulation of protein kinase activity by calcium and cAMP was investigated in cytosolic and particulate preparations from isolated mouse pancreatic acini. In cytosol, three protein kinase activities could be distinguished: a calcium-activated kinase activity that was increased by exogenous calmodulin (CaM) and abolished by treatment of cytosol with a phenothiazine-coupled resin, a calcium-activated kinase activity dependent on phosphatidylserine (PS), and cAMP-activated kinase activity. Phosphorylation of a Mr = 92,000 cytosolic protein was greatly increased by both CaM-dependent and cAMP-activated kinases, whereas PS-dependent kinase activity most heavily phosphorylated proteins of Mr = 62,000 and 40,000. In addition, these kinase activities demonstrated differences in specificity for exogenous protein substrates. CaM-and PS-dependent kinases were completely blocked by trifluoperazine; the inhibitor protein of cAMP-activated protein kinase selectively inhibited cAMP-activated kinase activity. Exogenous CaM decreased the concentration of free calcium for half-maximal activation of CaM-dependent kinase activity from 5.5 +/- 0.5 to 1.2 +/- 0.3 microM Ca2+; half-maximal activation of the PS-dependent and cAMP-activated kinase activities was achieved at 12 microM Ca2+ and 40-50 nM cAMP, respectively. In a particulate fraction depleted of endogenous CaM, CaM-dependent and cAMP-activated kinase activities were detected. In conclusion, this study demonstrates the existence of protein kinases in pancreatic acini which may be involved in the action of pancreatic regulatory agents that use calcium or cAMP as an intracellular messenger.

Beta-Adrenergic relaxation and cAMP kinase activation in coronary arterial smooth muscle.

If beta-adrenergic relaxation of smooth muscle is partly mediated by the adenosine 3',5'-cyclic monophosphate (cAMP) system, then beta-stimulation should be correlated to activation of cAMP-dependent protein kinase (cPK). Studies were performed with bovine coronary arterial strips to identify isozymic forms of cPK and to determine if beta-relaxation is correlated to activation of cPK (reflected by elevated ratios of cPK activity without cAMP to cPK activity with cAMP). Both ion exchange chromatography and a new electrophoretic technique revealed two cPK isozymes (types I and II). No change in cPK activity occurred in strips contracted with 30 mM KCl. In contrast, dose- and time-dependent relaxation during beta-stimulation with isoproterenol was highly correlated to parallel increases in cPK activity. Increased cPK activity was inhibited in assays performed with a specific inhibitor of cPK. Both relaxation and activation of cPK were abolished during beta-adrenergic blockade with propranolol. Relaxation by KCl removal or the ionophore R02-2985, unlike beta-mediated relaxation, did not increase cPK activity. These findings show that beta-mediated relaxation of isolated coronary arterial strips specifically activates cPK, and they support the hypothesis that beta-induced relaxation of vascular smooth muscle involves the cAMP system.

Multiple sites for interaction of prostaglandin and vasopressin in toad urinary bladder

The interaction of vasopressin with prostaglandins were examined in the toad bladder by determining water flows, cAMP levels, and cAMP-dependent protein kinase activity. Both water flow and activation of cAMP-kinase in response to vasopressin were enhanced after prostaglandin inhibition, consistent with inhibition of vasopressin-induced cAMP generation by endogenous prostaglandins. On the other hand exogeneous PGE stimulated cAMP generation. PGE1 (10(-7) M) alone did not increase water flow but activated kinase more than vasopressin only. Addition of PGE1 (10(-7) M) and vasopressin inhibited water flow as compared with vasopressin along but increased the kinase ratio above that with vasopressin only. PGE2 (10(-5) M) increased the cAMP content and kinase ratio even more than vasopressin but again resulted in no water flow. Addition of vasopressin and PGE2 (10(-5) M) increased water flow but did not alter cAMP content or the kinase ratio compared with PGE2 alone. Similar results were obtained with PGE1. Accordingly, prostaglandin dissociates cAMP levels and kinase ratio from the hydroosmotic response, suggesting that PGE2 inhibits steps distal to cAMP. Consistent with this, in bladders pretreated with naproxen or meclofenamate, PGE2 (10(-8) to 10(-6) M) inhibited the response to submaximal doses of cAMP (5 mM) or 8-bromo-cAMP (0.03 mM). Furthermore, pretreatment with naproxen significantly enhanced the response to cAMP (5 mM). These studies provide evidence for vasopressin-PGE interaction at the site of cAMP generation and also at a step(s) unrelated to cAMP generation.