Inhibition of serine-threonine protein phosphatases decreases barrier function of rat pulmonary microvascular endothelial cells.

The flux of multisized fluorescein-isothiocyanate-labeled hydroxy ethyl starch (FITC-HES) macromolecules was used to assess changes in barrier function of rat pulmonary microvascular endothelial cell (RPMVEC) monolayers exposed to protein phosphatase (PP) inhibitors or cGMP analogs and atriopeptin (ANF). Two potent PP inhibitors, calyculin A (CalA) and okadaic acid (OA), increased RPMVEC permeability in a dose- and time-dependent manner, and CalA had a higher intrinsic activity than OA. In contrast, ANF and potent cGMP analogs had no effect on basal RPMVEC permeability. The phosphohistone PP activity contained in RPMVEC sonicates was inhibited by OA with an inhibition profile that suggested at least two components were present, with PP2A accounting for approximately 70% of the OA-inhibitable phosphohistone phosphatase activity. Following separation with heparin-Sepharose chromatography, PP activity exhibited equipotent inhibition by CalA and differential inhibition by OA. Differential inhibition of PP1 and PP2A by OA suggested that PP1 is involved in regulating RPMVEC barrier function. Permeabilized RPMVEC showed increased phosphorylation of several proteins in the presence of phosphatase inhibitors. Treatment with KT 5926, a myosin light chain (MLC) kinase (MLCK) inhibitor, or rolipram, a phosphodiesterase inhibitor, decreased 32P incorporation into immunoprecipitated MLC by CalA and OA. However, this effect did not abolish either the CalA- or OA-induced decrease in the RPMVEC barrier function. Localization of filamentous (F) actin was at the periphery as well as in the cytoplasm and perinuclear region, whereas nonmuscle myosin was seen in the perinuclear region. Neither of these patterns was changed in the presence of CalA. Thus, cGMP does not alter RPMVEC permeability, but inhibition of PP activity results in loss of barrier function by a mechanism independent from MLC phosphorylation.

Cyclic GMP accumulation in pulmonary microvascular endothelial cells measured by intact cell prelabeling.

Cyclic GMP accumulation in cultured rat pulmonary microvascular endothelial cells (RPMVEC) was studied with a new prelabeling method developed using intact platelets and smooth muscle cells (1). [3H]-hypoxanthine was used to radiolabel the cellular guanine nucleotide pool. Neutral alumina and Dowex-50 double column chromatography was used to purify and quantitate the levels of [3H]-cyclic GMP. Changes in cyclic GMP metabolism in short and long term RPMVEC cultures were studied using rat atrial naturetic factor 8-33 (ANF) and sodium nitroprusside (SNP) in the presence and absence of cyclic nucleotide (CN) phosphodiesterase (PDE) inhibitors. In RPMVEC exogenous hypoxanthine was incorporated into both low (65% uptake) and high (34% uptake) passage cells in a time-dependent manner reaching maximum incorporation near 8 hours. Basal cyclic GMP values in both groups were 0.003% of the total cellular tritium (9 x 10(6) and 4 x 10(6) cpm/10(6) cells, respectively). ANF treatment of prelabeled RPMVEC resulted in a 10- to 12-fold increase in [3H]-cyclic GMP in the absence of CN PDE inhibitors (EC50 = 5.4 nM). However, incubation with SNP showed no changes in cellular cyclic GMP accumulation. Several relatively selective CN PDE inhibitors had no effect on ANF or SNP induced cyclic GMP accumulation in RPMVEC. The ANF induced cGMP accumulation was verified by radioimmunoassay. These studies confirm the utility of the hypoxanthine prelabeling technique to monitor intact microvascular EC cyclic GMP accumulation. Cultured RPMVEC show little or no functional soluble guanylate cyclase or cyclic GMP PDE activity.

Altered expression of cyclic nucleotide phosphodiesterase isozymes during culture of aortic endothelial cells.

Primary cultures of bovine aortic endothelial cells (BAEC) express cyclic nucleotide phosphodiesterase (CN PDE) isozymes of the PDE2, PDE4 and PDE5 gene families. We report here that the isozyme profiles of CN PDE and the amounts of each vary with the passage number of BAEC cultures. Characterization by anion-exchange chromatography and pharmacological criteria were used to study CN PDE in early (4-6), intermediate (6-10), and late (> 17) passages of purified BAEC. PDE2 and a minor fraction of PDE5 accounted for cyclic GMP hydrolysis in early passages, but both isozymes were lost with cell passage. Cyclic AMP was hydrolyzed by both PDE2 and PDE4 isozymes in early passage endothelial cells, but PDE4 was increased dramatically in higher passage cells. Also appearing in the higher passage cells were prominent PDE1 and minor PDE3 activities. The ratios of cytosolic to particulate activities were similar at all passages. BAEC PDE isoforms in intact cells assessed by [3H]-adenine prelabeling showed that atriopeptin II decreased isoproterenol-induced cyclic AMP accumulation in early but not later passage cells, consistent with the loss of PDE2 expression. Enhancement of isoproterenol-induced cyclic AMP accumulation by rolipram, a PDE4 inhibitor, was also greatly diminished during culture passages. Changes in CN PDE isoform expression and consequent cyclic AMP turnover validate the importance of considering cell passage number when cultures of BAEC are used to study the regulation of endothelial cell cyclic nucleotide metabolism and processes mediated by cyclic nucleotides in this model system.

Comparison of indolidan analog binding sites of drug antibody and sarcoplasmic reticulum with inhibition of cyclic AMP phosphodiesterase.

Dihydropyridazinone(DHP) derivatives such as indolidan are positive inotropic agents that show inhibition of cyclic AMP phosphodiesterase(PDE) activity. Indolidan inhibition is selective for PDE3 among the seven PDE gene families. DHP derivatives and related analogs have been used to define critical regions of the active site of PDE3 isoforms and radiolabeled analogs have been used to define indolidan sarcoplasmic reticulum (SR) receptor sites. We report here studies comparing the structure-activity relationships (SAR) for PDE3 inhibition with indolidan binding to two types of sites: canine SR and a monoclonal antibody derived against indolidan conjugated to a hemocyanin. SR and monoclonal antibody binding both fit singlesite, high affinity models (IC50 = 1.2 and 62 nM) that were near 52 and 360 times that of SR PDE3. Indolidan and thirteen analogs showed similar competition with either SR 3H-LY186126 binding or SR PDE3 inhibition. Antibody binding maintained selectivity but showed a different rank order potency for SR binding. Indole ring C3 methylation increased and DHP ring C4' methylation decreased indolidan monoclonal antibody binding while both substitutions increased SR binding. These studies support the hypothesis that SR PDE3 is a cardiotonic receptor site in myocardial membranes and indicate that models of the structural features of binding sites derived from inhibitor data alone could produce models with limited topography relative to the natural ligand.

Cyclic nucleotide phosphodiesterases and vascular smooth muscle.

At least 30 different phosphodiesterase (PDE) enzymes have now been identified in mammalian tissues and cells, many of which are products of separate genes. These different isoenzyme forms can be subdivided into seven families based on their genetic and functional characteristics. Relatively specific inhibitors are available for at least five of these PDE families. A functional classification based on substrate specificity, regulatory properties, and sensitivity to inhibition by isozyme- and tissue-selective inhibitors can be used in describing the PDEs of vascular smooth muscle. Inhibition of these PDEs, especially with inhibitors of the PDE3 isoform, promotes vascular relaxation, particularly if the preparation of smooth muscle has been preconracted. For the most part, the drugs appear to act directly on smooth muscle; their effects are usually observed in endothelium-denuded preparations. In addition to their cardiotonic properties, many PDE3 inhibitors possess antiplatelet and thrombolytic activities, thereby suggesting the potential benefit of these drugs in treating diseases of the cardiovascular system. Isozyme- and cell-specific drugs have been shown to alter the synthetic state (i.e. proliferative phenotype) of smooth muscle cultures toward the appearance of the contractile phenotype. This suggests the possible use of selective PDE inhibitors to minimize the problem of restenosis seen after angioplasty. The development of novel methods to deliver more potent and selective PDE inhibitors to individual cell types and subcellular locales will lead to new therapeutic uses for this class of drugs in diseases of the cardiovascular system.

Transcriptional regulation of secretogranin II and chromogranin B by cyclic AMP in a rat pheochromocytoma cell line.

When PC-12 cells were treated with 10 microM forskolin, the expression of two members of the granin family, secretogranin II (SgII) and chromogranin B (CgB), were differentially regulated. SgII mRNA levels declined progressively after forskolin treatment to reach a level of 22 +/- 1% of control after 48 hr, whereas CgB mRNA levels increased more rapidly, reaching a maximum of 3-fold above control after 24 hr. The dependence of these changes on an increase in cellular cAMP levels, activation of cAMP-dependent protein kinase (PKA), protein synthesis, and changes in the rate of transcription was investigated. The effects of forskolin on SgII and CgB mRNAs were reproduced by 1 mM 8-bromo-cAMP but not by 10 microM 1,9-dideoxyforskolin, an inactive analog of forskolin. The actions of forskolin on SgII and CgB mRNAs were blocked by treatment with 60 microM H-89, a selective PKA inhibitor, and were blunted in PKA-deficient PC-12 cell clones. To examine whether forskolin action was dependent on ongoing protein synthesis, PC-12 cells were treated with 1 microgram/ml cycloheximide before the addition of forskolin. The reduction in SgII mRNA levels by forskolin was not evident in PC-12 cells treated with cycloheximide. Rather, in the presence of cycloheximide, forskolin stimulated SgII mRNA levels 3.6 +/- 0.7-fold above control. The induction of CgB mRNA by forskolin was not affected by cycloheximide treatment. The superinduction of SgII mRNA by cycloheximide and forskolin was related to the extent of protein synthesis inhibition, was observed in cells treated with forskolin and other protein synthesis inhibitors, and was blunted in PKA-deficient PC-12 cells, suggesting that this effect was dependent on inhibition of protein synthesis and activation of PKA. To determine whether changes in SgII and CgB mRNA levels resulted from changes in the rate of transcription, nuclear run-on assays were performed in nuclei isolated from PC-12 cells that had been treated for 2 hr with cycloheximide, forskolin, or the two combined. Transcription of the SgII gene was not significantly affected by treatment with either forskolin or cycloheximide alone but was increased 12.9 +/- 1.0-fold above control in nuclei from cells treated with cycloheximide and forskolin together. Forskolin caused a 3.8 +/- 0.8-fold induction of CgB transcription.(ABSTRACT TRUNCATED AT 400 WORDS)

Reversal of pulmonary capillary ischemia-reperfusion injury by rolipram, a cAMP phosphodiesterase inhibitor.

Isoproterenol (ISO) and forskolin, agents that increase adenosine 3',5'-cyclic monophosphate (cAMP) via adenylyl cyclase activation, reverse lung injury associated with increased microvascular permeability. We studied the role of rolipram, a relatively isozyme-selective cAMP phosphodiesterase (PDE) inhibitor, in reversing increased capillary permeability due to ischemia-reperfusion (I/R), a form of oxidant injury in the lung, by using the isolated perfused rat lung model. Rolipram (2 microM) administered after 45 min of ischemia and 45 min of reperfusion reduced I/R-increased permeability as measured by the capillary filtration coefficient to control lung values. Computer image analysis of air space edema and perivascular cuffing, as well as wet-to-dry weight ratios, confirms the permeability reversal by rolipram administration. Rolipram inhibition of cAMP PDE in the lung was assessed by using [3H]adenine prelabeling adapted for the whole lung and perfusate [3H]cAMP accumulation. Rolipram failed to increase perfusate cAMP alone but dramatically increased perfusate cAMP above ISO alone. Dose-response relationships of ISO or rolipram show a close correlation of the half-maximal effective dose (ED50) for injury reversal and perfusate cAMP production. The combination of rolipram and ISO produced synergistic reversal of I/R injury. We conclude that reversal of I/R-induced increased microvascular permeability can be achieved with rolipram and that the mechanism of action of rolipram is probably through PDE isozyme-selective inhibition. The similarity of the ED50 values for cAMP efflux and reversal of permeability increases also supports a close coupling between cAMP accumulation and endothelial cell permeability.

Cyclic GMP-dependent protein kinase activity in rat pulmonary microvascular endothelial cells.

Cyclic GMP-dependent protein kinase (cGPK) activity was determined in rat pulmonary microvascular endothelial cells (RPMVEC) using cGMP-stimulated phosphorylation of BPDEtide and histone F2B substrates in the presence of PKI [peptide inhibitor of cAMP-dependent protein kinase (cAPK)]. RPMVEC cGPK activity was localized to the 100,000 x g cytosolic fraction. The EC50 for cGMP activation in the presence of PKI was 0.16 microM and H-89 inhibition under similar conditions showed an IC50 value of 0.16 microM. Anion-exchange chromatography of RPMVEC and rat lung cytosolic fractions showed separation of the cGMP-dependent from the cGMP-independent protein kinase activity and similar elution conductivities. Further, Western blots of RPMVEC active DEAE-Trisacryl fractions showed immunoreactivity using bovine Type I cGPK antiserum. Preliminary studies reveal six potential substrates phosphorylated by cGPK in RPMVEC. These studies describe an endothelial cell (EC) cGMP-receptor, cGPK, in addition to cGMP-activated (Type II) phosphodiesterase (PDE).

Effects of poloxamer 188 in a rabbit model of hemorrhagic shock.

Poloxamer 188 is a synthetic surfactant that reduces the viscosity of whole blood without hemodilution. It is postulated that poloxamer 188 would improve outcome if administered during retransfusion following hemorrhage. Rabbits were anesthetized and instrumented for 3 hours of hemodynamic monitoring. After stabilization, blood was withdrawn over a 5 minute period to reduce mean arterial pressure to 35 mmHg (4.7 kPa). Following a 60 minute shock period, animals were randomly assigned to 1 of 5 experimental groups (n = 8 in each): (1) Shock (no retransfusion); (2) Transfusion (retransfusion of autologous shed blood); (3) Volume (retransfusion with autologous blood and infusion of an additional volume of normal saline equivalent to the volume of poloxamer 188 given in the next 2 experimental groups); (4) Low and (5) High drug (i.v. bolus of 200 mg/kg of poloxamer 188 over 5 minutes at retransfusion, followed by a continuous infusion of poloxamer 188 at 50 mg/kg/hr in the Low drug group and 200 mg/kg/hr in the High drug group). All animals in a surgery Control group (n = 6) remained stable during the 3 hour monitoring period. In contrast, none of the animals in the Shock group remained alive, confirming this to be a relevant model of trauma and severe hemorrhagic shock. There were significantly more animals surviving at the end of the monitoring period in the two groups that received poloxamer 188 (numbers of animals alive after 3 hours = 7 of 8 in the High group and 6 of 8 in the Low group) compared to the Transfusion (4 of 8) and Volume (2 of 8) groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of acute and continuous administration of morphine on the cyclic AMP response induced by norepinephrine in rat brain slices.

Pre-incubated cortical brain slices from adult male Sprague Dawley rats when challenged by exogenous norepinephrine (NE) exhibited a dose-dependent increase in the level of endogenous cyclic 3',5' adenosine monophosphate (cyclic AMP), with the maximal response elicited at 50 microM NE concentration. The administration of 50 mg/kg sub-cutaneous (Sub-Q) morphine 5 minutes before sacrifice significantly increased the responsiveness of the brain slices to the NE-induced cyclic AMP response at 0.5, 5.0, and 50.0 microM NE. Sustained administration of morphine from the subcutaneously implanted morphine pellet (75 mg morphine base) attenuated the potentiated cyclic AMP response to NE in the brain slices of the rats exposed to a single challenge dose of 50 mg/kg (Sub-Q) morphine 5 minutes before sacrifice. This tolerance or attenuated response is first observed 24 hours after morphine pellet implantation with maximal tolerance observed at 48 hours after the pellet implantation. A complete reversal of attenuated NE-induced cyclic AMP response was observed when the 3 day morphine implanted rats were injected with a challenge dose of naloxone (4 mg/kg, Sub-Q) at 10 minutes prior to the acute administration of 50 mg/kg Sub-Q injection of morphine 5 minutes before sacrifice. These results suggest that both acute and prolonged administration of morphine alters NE-induced cyclic AMP response of the brain slices, and that naloxone, an opioid antagonist, reverses this response. This is perhaps due to morphine-induced alterations in the availability of NE in the CNS.

Inotropic responses to selective (RO 20-1724 and SQ 65,442) and nonselective (trequinsin) inhibitors of cyclic AMP-specific class IV phosphodiesterase in newborn, immature, and adult rabbit myocardium.

In contrast to myocardium from adult rabbits, myocardium from newborns is insensitive to the inotropic effects of selective inhibitors (e.g. amrinone, milrinone, and indolidan) of the cGMP-inhibited high-affinity cAMP phosphodiesterase (PDE) localized in the sarcoplasmic reticulum. This difference may be explained at least partially by our recent observation that this cAMP PDE activity is low in sarcoplasmic reticulum from newborns. Furthermore, because the predominant cytosolic high-affinity cAMP PDE activity in newborns is a cGMP-insensitive form, we postulated that selective inhibitors of this form of cAMP-specific PDE may increase cardiac contractility in newborns. Therefore, the inotropic effects of RO 20-1724 and SQ 65,442 (selective inhibitors of cGMP-insensitive, high-affinity cAMP PDE) were compared with trequinsin (a potent, less selective PDE inhibitor) in right ventricular papillary muscles isolated from newborn (NB; 24-48 h), immature (14-16 d), and adult New Zealand White rabbits. At a drug concentration of 100 microns, RO 20-1724 and SQ 65,442 depressed maximal rate of tension development to 67 +/- 4 and 70 +/- 2% of control, respectively, in NB papillary muscles. The NB response to RO 20-1724 differed significantly from the immature (127 +/- 2%) and adult (115 +/- 3%) groups (p less than 0.05), but the effects of SQ 65,442 were comparable among the three age groups. In contrast, trequinsin exerted a positive inotropic effect in the NB group (355 +/- 22% of control) that was substantially greater than the maximal response obtained in the immature (139 +/- 6% of control) or adult (131 +/- 5% of control) groups (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Role of cAMP-dependent protein kinase in cAMP-mediated vasodilation.

In this study, the role of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase A (PKA) in cAMP-dependent relaxation was assessed in the isolated-perfused rat lung using a PKA inhibitor, Rp-cAMPS, 8-bromo-cAMP (8-BrcAMP), and the diterpene activator of adenylate cyclase (AC), forskolin (FSK). A role for K+ channels was also assessed with the nonselective K+ channel blocker, tetraethylammonium (TEA, 10 mM), and an ATP-sensitive K+ channel inhibitor, glibenclamide (GLI, 100 microM). Both 8-BrcAMP (0.1-1.0 mM) and RSK (0.1-10 microM) dose-dependently attenuated the peak pressor response to alveolar hypoxia (HPR). Rp-cAMPS potentiated the HPR and attenuated 8-BrcAMP-mediated vasodilation but had no effect on FSK-mediated vasodilation. FSK-mediated vasodilation was not mimicked by 1,9-dideoxy-FSK, which is biologically inactive on AC but alters K+ channels identically to FSK, nor was it attenuated by the platelet-activating factor antagonist SRI 63-441 or the cyclooxygenase inhibitor indomethacin. TEA, but not GLI, attenuated FSK-mediated vasodilation. Similarly, TEA attenuated 8-BrcAMP-mediated vasodilation. These results support roles for PKA and indirect gating of a non-ATP-sensitive K+ channel in mediating cAMP-dependent pulmonary vasodilation.

Effects of hemorrhagic shock and retransfusion on catalase and superoxide dismutase activities in rabbits.

The effects of hemorrhagic shock and retransfusion on the activities of two endogenous antioxidant enzymes, catalase and superoxide dismutase, were studied in male New Zealand white rabbits. Following instrumentation and stabilization, blood was withdrawn to reduce mean arterial pressure to 35 mmHg (SHOCK). After 60 min of shock, the warmed autologous blood was infused and the animals were allowed to recover for an additional 30 min (RETRANS). CONTROL animals were instrumented but not bled or transfused. A 60 min period of hemorrhagic shock had no significant effect on tissue or plasma activities of catalase or superoxide dismutase. However, transfusion following hemorrhagic shock resulted in an increase in catalase activity of nearly two-fold in the liver and five-fold in the small intestine. In contrast, superoxide dismutase activity remained unchanged after retransfusion in other tissues and in plasma. These results indicate that superoxide dismutase and catalase activities may be preserved or even induced following transfusion for hemorrhagic shock.

Activation of rabbit liver high affinity cAMP (type IV) phosphodiesterase by a vanadyl-glutathione complex. Characterization of the role of the sulfhydryl.

Activation of rabbit liver microsomal high affinity cAMP phosphodiesterase (Type IV PDE) by vanadyl-glutathione complexes was studied as a possible model of insulin stimulation of the enzyme in a cell-free system. The effect of VO.2GSH activation of PDE was a 21-fold decrease in the IC50 value for cGMP inhibition and a 2.6-fold increase in the Vmax of the higher affinity cAMP catalytic site. Cyclic AMP and cGMP substrate affinities and cGMP hydrolysis were unaffected by VO.2GSH activation. Selective Type IV PDE inhibitors and cGMP analogs indicated that VO.2GSH complexes activated the cGMP-inhibitable form of the Type IV PDE activities which co-localized in hepatic microsomes. The Type IV PDE activating complex appears to consist minimally of vanadyl ion and 2 oxidized electron donor compounds. The components of the electron donor required to achieve an enzyme activation complex are: 1) a free -SH group as the electron donor for vanadate reduction and 2) a minimum structure of cysteamine (NH2-CH2-CH2-SH). Maximal activation of the enzyme required near 2:1 molar ratios of either glutathione or cysteamine mixed with sodium orthovanadate. Active vanadyl-cysteamine complexes were isolated by reverse- phase high performance liquid chromatography. Tungsten, niobium, and tantalum, but not manganese, chromium, or molybdenum, substituted for vanadium to form enzyme-activating complexes with glutathione. VO.RSH complex activation occurred rapidly upon addition to microsomes and was reversible. We conclude from these studies that VO.RSH complexes and insulin activate the same form of Type IV PDE in rabbit liver microsomes; our findings are discussed with respect to the involvement of a possible electron transfer enzyme oxidation in the activation mechanism.

Selective inhibition of cGMP-inhibitable cAMP phosphodiesterase decreases pulmonary vasoreactivity.

Guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate (cAMP) are mediators of smooth muscle relaxation. In this study, selective inhibitors of phosphodiesterase (PDE) isozymes were used to assess the role of cyclic nucleotide hydrolysis in angiotensin II (ANG II) and hypoxic pulmonary vasoconstriction. In isolated rat lungs, the hypoxic pressor response (HPR) was induced with a 95% N2-5% CO2 gas mixture. When administered during the plateau of the HPR, trequinsin (nonselective PDE inhibitor) and indolidan (cGMP-inhibitable cAMP PDE inhibitor) significantly (P = 0.01) decreased the pulmonary arterial pressure (Ppa) by 60 +/- 7 and 53 +/- 3%, respectively, compared with zaprinast (cGMP PDE inhibitor), rolipram (cGMP-insensitive cAMP PDE inhibitor), and the 0.1% dimethyl sulfoxide (DMSO) vehicle control, which decreased the Ppa by 6 +/- 3, 4 +/- 3, and 0%, respectively. In the trequinsin and indolidan groups, the subsequent ANG II pressor responses and HPRs were significantly (P = 0.01) decreased when compared with the zaprinast, rolipram, and DMSO groups. During normoxia, none of the PDE inhibitor (0.3-30 microM) had an effect on the baseline Ppa. These results suggest that cAMP hydrolysis by the cGMP-inhibitable cAMP PDE play a significant role in pulmonary vascular tone regulation.

Phosphodiesterase II, the cGMP-activatable cyclic nucleotide phosphodiesterase, regulates cyclic AMP metabolism in PC12 cells.

Analysis of cyclic nucleotide phosphodiesterase (PDE) activity in cellular fractions from cultured rat pheochromocytoma (PC12) cells has shown that the predominant hydrolytic activity in both cytosolic and particulate compartments is characteristic of a PDE II, the cGMP-activatable family of PDE isozymes. Cytosolic PDE activity was purified to a high degree utilizing DE-52 anion exchange and cGMP-Sepharose affinity chromatographies. The physicochemical properties of PC12 PDE II were similar to those of PDE II isolated from particulate or soluble fractions of other tissues, including subunit molecular weight of approximately 102,000, activation of cAMP hydrolysis by cGMP, and positive cooperative kinetic behavior for cAMP and cGMP hydrolysis. The potential role of PDE II in regulating cAMP metabolism in intact PC12 cells was studied using an [3H]adenine prelabeling technique. Stimulation of PC12 cell adenosine receptors resulted in a 5-8-fold increase in cAMP accumulation. Removal of the adenosine stimulus by the addition of exogenous adenosine deaminase resulted in a rapid decay of cAMP to prestimulated basal levels within 2 min. Treatment of PC12 cells with atrial natriuretic factor or sodium nitroprusside caused 1) increased intracellular cGMP levels, 2) attenuation of adenosine-stimulated cAMP accumulation, and 3) increased rates of cAMP decay after removal of the adenosine stimulus. Treatment of PC12 cells with HL-725 (a potent inhibitor of isolated PDE II activity in vitro) caused 1) increased basal cAMP accumulation, 2) potentiation of adenosine-stimulated cAMP accumulation, and 3) retardation of the rate of cAMP decay after removal of the adenosine stimulus. HL-725 blocked both the attenuation of cAMP accumulation and the accelerated rate of cAMP decay observed with the cGMP-elevating agents. These results suggest that, in PC12 cells, drugs or hormones that inhibit PDE II or increase intracellular cGMP levels to activate PDE II can modulate cAMP metabolism by altering the catalytic status of the enzyme.

Fura 2 analysis of cytosolic calcium regulation in elutriated rat gastric parietal cells.

The calcium probe, Fura 2, is used to establish and partially characterize histamine-, carbachol-, and forskolin-induced calcium transients in enriched parietal cell populations prepared by centrifugal elutriation of dispersed rat fundic mucosa cell isolates. The magnitude of the maximal carbachol response, which is blocked by atropine but not cimetidine, is nearly five times that of histamine or forskolin. Time to peak responses for carbachol, forskolin, and histamine are approximately 7, 17, and 28 sec, respectively. Carbachol-, histamine-, and forskolin-induced increases in Fura 2 fluorescence appear dependent upon extracellular calcium, since these responses are attenuated in low calcium media and blocked by EGTA in low-calcium media or by lanthanum in high- or low-calcium medium. Trifluoperazine and fenoctimine, at concentrations that inhibit secretion, have no effect on either carbachol- or histamine-induced increases in cytosolic calcium. Seven major calcium/EGTA-sensitive phosphoproteins are identified by SDS-PAGE electrophoresis of ATP 32P-labeled cell sonicates. We conclude that cytosolic calcium in enriched rat gastric parietal cell populations is regulated by secretagogue receptor-controlled calcium channels. We postulate that these channels may be controlled by cyclic AMP-dependent phosphorylation, since neither changes in cyclic AMP nor calcium alone mediate the effects of secretagogues entirely, but the interplay between these two second-messenger systems potentiates the actions of these agents. The role of cytosolic calcium as a second messenger in secretagogue action appears similar to that of cyclic AMP in that a specific cellular concentration must be reached to initiate acid secretion.

Subcellular distribution of high-affinity type IV cyclic AMP phosphodiesterase activities in rabbit ventricular myocardium: relations to post-natal maturation.

Cytosolic and particulate Type IV (high-affinity) cAMP phosphodiesterase (PDE) activities were isolated from the ventricular myocardium of newborn (NB; 24 to 48 h), immature (IM; 14 to 16 days) and adult (AD; 6 to 8 months) rabbits. Cytosolic activity from each age group was resolved into three distinct peaks of activity by DEAE cellulose anion exchange chromatography. Type IV PDE activity was identified as a predominant activity in the cytosolic peak III activity in all three age groups when measured with 0.25 microM cAMP as substrate. A particulate Type IV PDE activity was associated with the sarcoplasmic reticulum (SR) fractions in each age group. No significant age-related changes in the affinity of the particulate enzyme for cAMP (apparent Km = 0.3 to 0.5 microM) were evident, but the Vmax for this SR-associated activity increased from 553 +/- 7 pmol/min/mg in the NB to 725 +/- 9 pmol/min/mg in the IM and 2450 +/- 33 pmol/min/mg in the AD. In each age group, milrinone, imazodan, piroximone and indolidan were more potent inhibitors of the SR-associated activity as compared with the cytosolic peak III activity. In contrast, RO 20-1724 and rolipram were relatively more selective inhibitors of the cytosolic peak III activity. Age-related differences in the sensitivity of type IV PDE to inhibition was dependent upon the selectivity of the inhibitor and the subcellular enzymic distribution. Cytosolic peak III PDE activity was further resolved by gel filtration chromatography into two peaks. Hydrolysis of cAMP by the higher molecular weight peak was inhibitable by cGMP (IC50 = 0.25 +/- 0.07 microM in NB and 0.07 +/- 0.01 microM in AD) whereas the lower molecular weight peak activity was relatively insensitive to inhibition by cGMP (IC50 greater than 100 microM). The lower molecular weight peak constituted a relatively greater proportion of the total peak III activity in the NB as compared to the AD. Analysis of the kinetics of cGMP inhibition of high-affinity cAMP hydrolysis was consistent with the presence of a greater number of high-affinity (presumably drug-sensitive) binding sites in the SR-associated activity as compared to the cytosolic peak III activity in both NB and AD. These results support the hypothesis that the cGMP-inhibitable Type IV PDE activity may be the primary site of action for certain newer cardiotonic drugs. Differences in drug action in young versus adult myocardium may be related to the selectivity of the cardiotonic drugs for this specific isozyme and its lower specific activity during the early stages of maturation.

Inotropic responses to cyclic nucleotide phosphodiesterase inhibitors in immature and adult rabbit myocardium.

Contractile dose-response relationships for amrinone, milrinone, piroximone, and sulmazole were compared in right ventricular papillary muscles isolated from adult and 14-16-day-old immature rabbits. These drugs were effective inotropic agents in immature myocardium as evidenced by substantial increases in the maximal rate of tension development. The rank order of maximum inotropic effect in the immature muscles was milrinone = sulmazole greater than piroximone greater than amrinone. Compared with adults, the relative magnitude of the inotropic response for each drug was greater in immature myocardium. Profiles of ventricular cytosolic cyclic nucleotide phosphodiesterase (PDE) activities resolved by anion exchange chromatography were similar for the two age groups. Immature myocardium was found to contain the Type IV (Peak III) high-affinity cAMP PDE that has been implicated in the mechanism of action of these drugs. Partially purified cytosolic Type IV PDE from immature and adult myocardium exhibited similar kinetic characteristics (cAMP Km = 0.9 microM; Vmax = 17 nmol/min/mg) and sensitivity to inhibition by cGMP. Except for piroximone, the inhibitory effect of each drug on cytosolic Type IV PDE activity from immature myocardium did not differ from the adult, as indexed by comparable concentrations required to inhibit activity by 50% (IC50) and Ki values (piroximone IC50 and Ki values were higher in the immature compared with the adult group). Thus, these studies demonstrated significant age-related differences in the contractile responses to amrinone, milrinone, piroximone, and sulmazole. These differences are not attributable to differences in myocardial cytosolic Type IV high-affinity cAMP PDE activity.(ABSTRACT TRUNCATED AT 250 WORDS)