Multidrug resistance-associated protein 4 regulates cAMP-dependent signaling pathways and controls human and rat SMC proliferation

The second messengers cAMP and cGMP can be degraded by specific members of the phosphodiesterase superfamily or by active efflux transporters, namely the multidrug resistance-associated proteins (MRPs) MRP4 and MRP5. To determine the role of MRP4 and MRP5 in cell signaling, we studied arterial SMCs, in which the effects of cyclic nucleotide levels on SMC proliferation have been well established. We found that MRP4, but not MRP5, was upregulated during proliferation of isolated human coronary artery SMCs and following injury of rat carotid arteries in vivo. MRP4 inhibition significantly increased intracellular cAMP and cGMP levels and was sufficient to block proliferation and to prevent neointimal growth in injured rat carotid arteries. The antiproliferative effect of MRP4 inhibition was related to PKA/CREB pathway activation. Here we provide what we believe to be the first evidence that MRP4 acts as an independent endogenous regulator of intracellular cyclic nucleotide levels and as a mediator of cAMP-dependent signal transduction to the nucleus. We also identify MRP4 inhibition as a potentially new way of preventing abnormal VSMC proliferation.     

Hydrolysis of N-methyl-D-aspartate receptor-stimulated cAMP and cGMP by PDE4 and PDE2 phosphodiesterases in primary neuronal cultures of rat cerebral cortex and hippocampus

Stimulation of N-methyl-D-aspartate (NMDA) receptors on neurons activates both cAMP and cGMP signaling pathways. Experiments were carried out to determine which phosphodiesterase (PDE) families are involved in the hydrolysis of the cyclic nucleotides formed via this mechanism, using primary neuronal cultures prepared from rat cerebral cortex and hippocampus. The nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) potentiated the ability of NMDA to increase cAMP and cGMP. However, among the family-selective inhibitors, only the PDE4 inhibitor rolipram enhanced the ability of NMDA to increase cAMP in the neurons. In contrast, only the PDE2 inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) enhanced the ability of NMDA to increase cGMP. Neither adenosine nor an adenosine deaminase inhibitor mimicked the effect of EHNA; this suggests that EHNA's inhibition of PDE2, not its effects on adenosine metabolism, mediates its effects on NMDA-stimulated cGMP concentrations. The PDE inhibitor-augmented effects of NMDA on cAMP and cGMP formation were antagonized by 5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine maleate (MK-801), verifying NMDA receptor mediation. In contrast, only NMDA-mediated cGMP formation was affected by altering either nitric oxide signaling or guanylyl cyclase; this suggests that NMDA-induced changes in cAMP are not secondary to altered cGMP concentrations. Overall, the present findings indicate that cAMP and cGMP formed in neurons as a result of NMDA receptor stimulation are hydrolyzed by PDE4 and PDE2, respectively. Selective inhibitors of the two PDE families will differentially affect the functional consequences of activation of these two signaling pathways by NMDA receptor stimulation.     

Stimulation of beta adrenoceptors in a human monocyte cell line (U937) up-regulates cyclic AMP-specific phosphodiesterase activity.

Experiments were conducted using undifferentiated U937 cells, a human monocytic cell line, to establish an in vitro model to examine the hormonal regulation of the cyclic AMP (cAMP)-specific phosphodiesterase (PDE IV). Standard chromatographic techniques, coupled with the use of inhibitors and activators that are selective for various phosphodiesterase (PDE) isozymes, were used to establish the PDE isozyme profile in supernatant fractions of U937 cells. When PDE activity was assessed using 1 microM [3H]cAMP as a substrate, 70 to 90% of the total U937 cell supernatant activity in the major peak eluting from anion-exchange columns was inhibited by 30 microM rolipram, a selective inhibitor of PDE IV. The remaining activity was nearly abolished by 10 microM siguazodan or 10 microM cyclic GMP (cGMP,) selective inhibitors of the cGMP-inhibited PDE. Kinetic analyses of the enzyme activity contained within this major peak of PDE activity revealed a cAMP Km = 3 microM and a rolipram Ki = 0.5 microM, values characteristic of PDE IV. Additional studies revealed the presence of a small amount of Ca++/calmodulin-stimulated PDE, but no cGMP-stimulated PDE or cGMP-specific PDE activity. In an effort to induce PDE activity in intact U937 cells by producing a sustained increase in cAMP content, cells were treated for 4 hr with salbutamol (1 microM), rolipram (30 microM) or a combination of both agents. The combination of salbutamol and rolipram produced a 2- to 3-fold increase in PDE activity in U937 cells; when used alone, rolipram was without effect whereas salbutamol induced an increase that was approximately one-half of that observed with the combination. Isozyme isolation and characterization revealed that the overall elevation of cellular PDE activity could be accounted for by a 2- to 3-fold increase in the Vmax of PDE IV with no change in its Km. The induction of PDE IV by salbutamol was: 1) concentration- and time-dependent; 2) detectable only after prolonged (2-4 hr) agonist exposure; 3) preceded by an increase in cAMP content and an activation of cAMP-dependent protein kinase; 4) mimicked by 8-bromo-cAMP and prostaglandin E2; 5) reversible within 3 hr of salbutamol removal; and 6) abolished by cycloheximide or actinomycin D. Collectively, these results indicate that the major PDE isozyme in the soluble fraction of U937 cells is PDE IV and that the activity of this enzyme is increased markedly in cells after prolonged exposure to agents that increase cAMP content.     

Inhibition of the low Km cyclic AMP phosphodiesterase and activation of the cyclic AMP system in vascular smooth muscle by milrinone

The purposes of the present study were to quantitate the effects of the cardiotonic/vasodilator milrinone on phosphodiesterase (PDE) isozymes isolated from vascular (aortic) smooth muscle from several species, and to quantitate changes in cellular cyclic AMP (cAMP) content, activation of cAMP protein kinase (cAPK) and vasorelaxation by milrinone in isolated guinea pig aortic smooth muscle. With PDE isozymes isolated from rat (Wistar-Kyoto or spontaneously hypertensive rats), guinea pig, monkey or canine aortic smooth muscle, milrinone is a potent (IC50 = 0.16-0.90 microM) and selective (100 times peak III relative to peak I) peak III inhibitor. The potency of milrinone and other vascular peak III PDE inhibitors parallels their potency as vasorelaxants in isolated guinea pig aortic rings (r = 0.86; P less than .01). Vasorelaxation of phenylephrine-contracted (3 microM) guinea pig aortic rings is accompanied by significant increases in cAMP content or cAPK activation with concentrations of milrinone greater than or equal to 10 microM. Temporally, significant increases in cAMP content accompany significant vasorelaxation; however, activation of cAPK is not significantly increased until at least 1 to 2 min after addition of milrinone. Similar concentration and temporal relationships are seen with the cAMP-related vasorelaxants papaverine and forskolin. As with milrinone, a temporal dissociation between increases in cAMP content and increases in cAPK activity ratio is evident.(ABSTRACT TRUNCATED AT 250 WORDS)     

Compound C inhibits vascular smooth muscle cell proliferation and migration in an AMP-activated protein kinase-independent fashion

6-[4-(2-Piperidin-1-yl-ethoxy)-phenyl]-3-pyridin-4-yl-pyrazolo[1,5-a] pyrimidine (compound C) is a cell-permeable pyrrazolopyrimidine derivative that acts as a potent inhibitor of AMP-activated protein kinase (AMPK). Although compound C is often used to determine the role of AMPK in various physiological processes, it also evokes AMPK-independent actions. In the present study, we investigated whether compound C influences vascular smooth muscle cell (SMC) function through the AMPK pathway. Treatment of rat aortic SMCs with compound C (0.02-10 μM) inhibited vascular SMC proliferation and migration in a concentration-dependent fashion. These actions of compound C were not mimicked or affected by silencing AMPKα expression or infecting SMCs with an adenovirus expressing a dominant-negative mutant of AMPK. In contrast, the pharmacological activator of AMPK 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside inhibited the proliferation and migration of SMCs in a manner that was strictly dependent on AMPK activity. Flow cytometry experiments revealed that compound C arrested SMCs in the G(0)/G(1) phase of the cell cycle, and this was associated with a decrease in cyclin D1 and cyclin A protein expression and retinoblastoma protein phosphorylation and an increase in p21 protein expression. Finally, local perivascular delivery of compound C immediately after balloon injury of rat carotid arteries markedly attenuated neointima formation. These studies identify compound C as a novel AMPK-independent regulator of vascular SMC function that exerts inhibitory effects on SMC proliferation and migration and neointima formation after arterial injury. Compound C represents a potentially new therapeutic agent in treating and preventing occlusive vascular disease.     

Aging and arteriosclerosis. The increased proliferation of arterial smooth muscle cells isolated from old rats is associated with increased platelet-derived growth factor-like activity

In vivo studies have suggested that the aorta from an old animal responds to injury with an exaggerated proliferation of smooth muscle cells (SMCs) compared with the response of this aorta from a young animal. In this study we compared proliferation of SMCs derived from uninjured old (less than 19 mo) and young (3-4 mo) rat aortas. Old SMCs grew more rapidly than young SMCs in the presence of medium containing competence factors (10% FCS or platelet-derived growth factor [PDGF]) as well as in their absence (2% PDS or serum-free media) as determined both by a short-term thymidine incorporation assay and by cell counts. Lysates prepared from old SMCs that had been grown in the absence of serum or PDGF stimulated proliferation of target cells more than lysates prepared from young SMCs; the effect was inversely related to cell density of the SMCs. This stimulatory effect of lysates was completely blocked by antibody to PDGF. After the growth-promoting activity of lysates was eliminated by anti-PDGF, growth-inhibiting activity was revealed. Lysates prepared from old SMCs had significantly less capacity to inhibit target cell growth. In the presence of exogenous heparin both the serum- or PDGF-stimulated proliferation and serum-free proliferation of old SMCs were decreased to the level of proliferation of young SMCs. These results suggest that the balance between growth-promoting and growth-inhibiting factors is altered in SMCs from old rats. This may contribute to the increased proliferative capacity of these cells in culture and may facilitate the development of atherosclerosis with age.     

Inhibition of rat vascular smooth muscle proliferation in vitro and in vivo by bone morphogenetic protein-2.

Vascular proliferative disorders are characterized by the proliferation of vascular smooth muscle cells (SMCs) and excessive extracellular matrix synthesis. We found that bone morphogenetic protein-2 (BMP-2) inhibited serum-stimulated increases in DNA synthesis and cell number of cultured rat arterial SMCs in a fashion quite different from that in the case of transforming growth factor-beta1 (TGF-beta1). In addition, TGF-beta1 stimulated collagen synthesis in SMCs, whereas BMP-2 did not. In an in vivo rat carotid artery balloon injury model, the adenovirus-mediated transfer of the BMP-2 gene inhibited injury-induced intimal hyperplasia. These results indicate that BMP-2 has the ability to inhibit SMC proliferation without stimulating extracellular matrix synthesis, and suggest the possibility of therapeutic application of BMP-2 for the prevention of vascular proliferative disorders.     

Insulin activation of cyclic AMP phosphodiesterase in intact ureteral segments

Low doses of insulin (0.1-50 nM) when presented to intact ureteral segments increase cyclic AMP (cAMP) phosphodiesterase (PDE) activity in subsequently isolated supernatant and particulate fractions. The stimulation of cAMP PDE occurs within 5 to 10 min of the introduction of insulin. When cyclic GMP or high concentrations of cAMP (greater than 5 microM) are used as substrate, insulin does not increase PDE activity. Although the insulin-increased cAMP PDE exhibits the same sensitivity as control PDE from untreated preparations to isobutylmethyl xanthine, a nonspecific PDE inhibitor, and M & B 22,948, a relatively selective cyclic GMP PDE inhibitor, differences in the degree of inhibition of PDE activity are seen in the insulin-treated and untreated preparations with the low Km cAMP PDE inhibitors Ro20-1724, rolipram, amrinone and milrinone and with cyclic GMP. Pertussis toxin, which modifies GTP regulatory proteins of the adenylate cyclase enzyme and the photoreceptor PDE, blocks cAMP PDE activation by insulin.     

Hepatoma-derived growth factor stimulates smooth muscle cell growth and is expressed in vascular development

Hepatoma-derived growth factor (HDGF) is the first member identified of a new family of secreted heparin-binding growth factors highly expressed in the fetal aorta. The biologic role of HDGF in vascular growth is unknown. Here, we demonstrate that HDGF mRNA is expressed in smooth muscle cells (SMCs), most prominently in proliferating SMCs, 8-24 hours after serum stimulation. Exogenous HDGF and endogenous overexpression of HDGF stimulated a significant increase in SMC number and DNA synthesis. Rat aortic SMCs transfected with a hemagglutinin-epitope-tagged rat HDGF cDNA contain HA-HDGF in their nuclei during S-phase. We also detected native HDGF in nuclei of cultured SMCs, of SMCs and endothelial cells from 19-day fetal (but not in the adult) rat aorta, of SMCs proximal to abdominal aortic constriction in adult rats, and of SMCs in the neointima formed after endothelial denudation of the rat common carotid artery. Moreover, HDGF colocalizes with the proliferating cell nuclear antigen (PCNA) in SMCs in human atherosclerotic carotid arteries, suggesting that HDGF helps regulate SMC growth during development and in response to vascular injury.     

Mechanisms of focal calcification in rabbit aortas: role of calcifying vesicles.

BACKGROUND: We previously demonstrated that calcification in rabbit thoracic aortas is initiated in the lower zone of the extensively thickened plaques (LZP) adjacent to the media. Whether osteogenesis or a local increase in calcifying vesicles underlies the focal calcification remains to be established. METHODS: To determine that focal calcification is related to osteogenesis, an obligatory osteogenic biomarker, alkaline phosphatase (ALP) activity, was evaluated in the unfixed thoracic aorta sections of rabbits fed a supplemental cholesterol diet and of humans with advanced atherosclerosis. To determine whether blood shortage to the smooth muscle cells (SMCs) imposed by intima thickening may increase calcifying vesicles, the effect of serum deprivation on the biogenesis of calcifying vesicles in cultured SMCs was investigated. RESULTS: (1) In contrast to positive rabbit kidney cross sections and consistent with the activity in various isolated subcellular fractions of aortas, ALP activity was absent in the media, adventitia, lesions, and LZP of rabbit aortas or in the fibrointima of human aortas. (2) Histologic assessments of the lesions indicate the absence of bone cells or osteoid. (3) Depletion of the serum from culture media caused a twofold increase in the levels of ALP-deficient and adenosine triphosphatase-rich calcifying vesicles, which were released from the cells by treatment with 0.05% trypsin-0.02% ethylenediaminetetraacetic acid for 15 minutes. CONCLUSIONS: (1) Focal calcification in rabbit aortas and diffuse mineralization in human fibrointima are not associated with osteogenesis, and (2) blockade of the blood supply to SMCs may trigger the cells to produce more calcifying vesicles, thereby leading to site-specific calcification in rabbit aortas.     

Nephron distribution of total low Km cyclic AMP phosphodiesterase in mouse, rat and rabbit kidney

The activity of cAMP degradation enzyme, cAMP phosphodiesterase (cAMP PDE), in renal tubules is a critically important factor in determining cellular cAMP levels, particularly in response to hormones. In this study we examine the nephron distribution of cAMP PDE activity in the mouse, rat and rabbit kidney and important cellular regulators of cAMP PDE, namely calmodulin and adenosine triphosphate (ATP). We assayed total low Km cAMP PDE in microdissected tubule segments, using 10(-6) M (3H) cAMP as a substrate. Activities were expressed in fentomol cAMP hydrolyzed per minute per mm tubular length or per one glomerulus. The content of ATP was measured in outer medullary collecting duct and medullary thick ascending limb of Henle's loop with microbioluminescence assay using firefly luciferase. In mouse kidney, cAMP PDE was significantly higher in all tubular segments compared to glomerulus. Proximal convoluted tubule, proximal straight tubule, medullary thick ascending limb of Henle's loop (mTAL), and outer medullary collecting duct (OMCD) had intermediated activity. Greater cAMP PDE activity was detected in cortical ascending limb of Henle's loop (cTAL), cortical collecting duct and in distal convoluted tubule (DCT). The highest activity was found in connecting tubules. In rat, nephron distribution of cAMP PDE activities was similar to mouse, except that activity in glomeruli was higher than in mouse glomeruli. In rabbit, nephron distribution of cAMP PDE activities was different from those of mouse and rat. There was no single prominent segment with high cAMP PDE activity. DCT and cTAL showed low enzyme activity. Overall, the highest cAMP PDE activities were measured in the mouse and the lowest were measured in the rabbit nephrons, with those of rat nephron showing an intermediate activity. The maximum effective dose of the calmodulin antagonist, trifluoperazine (200 microM), inhibited cAMP PDE in all nephron segments from the rat kidney. However, there is no statistical significance of its inhibition among nephron segments. In OMCD and mTAL of the rat kidney, cAMP PDE activity was inhibited by ATP (5 mM to approximately 10 mM) which is far beyond the physiological concentartion of ATP in normal epithelial cell. Actual determinations of ATP in mTAL and OMCD were 0.1 mM and 0.17 mM, respectively. These observations show that distal segments of tubules have more active catabolism of cAMP than proximal segments. cAMP PDE in each nephron segment appear to be almost equally dependent on trifluoperazine-sensitive pathway that may reflect the Ca2+-calmodulin system. Cellular concentration of ATP might not be involved in the regulation of the total low Km cAMP PDE activity in rat mTAL and OMCD.     

The mitogen-activated protein kinase pathway can mediate growth inhibition and proliferation in smooth muscle cells. Dependence on the availability of downstream targets.

Activation of the classical mitogen-activated protein kinase (MAPK) pathway leads to proliferation of many cell types. Accordingly, an inhibitor of MAPK kinase, PD 098059, inhibits PDGF-induced proliferation of human arterial smooth muscle cells (SMCs) that do not secrete growth-inhibitory PGs such as PGE2. In striking contrast, in SMCs that express the inducible form of cyclooxygenase (COX-2), activation of MAPK serves as a negative regulator of proliferation. In these cells, PDGF-induced MAPK activation leads to cytosolic phospholipase A2 activation, PGE2 release, and subsequent activation of the cAMP-dependent protein kinase (PKA), which acts as a strong inhibitor of SMC proliferation. Inhibition of either MAPK kinase signaling or of COX-2 in these cells releases them from the influence of the growth-inhibitory PGs and results in the subsequent cell cycle traverse and proliferation. Thus, the MAPK pathway mediates either proliferation or growth inhibition in human arterial SMCs depending on the availability of specific downstream enzyme targets.     

Phosphatidylethanolamine methyltransferase and cAMP, cGMP phosphodiesterases in lymphocytes and monocytes in sarcoidosis

Among the various hypotheses proposed to explain immune cell defect in sarcoidosis, we examined thoroughly that of Faguet who described abnormalities of signal transmission at lymphocyte membrane level. Phosphatidylethanolamine methyltransferase and cAMP cGMP phosphodiesterases were studied in blood lymphocytes and monocytes from 8 subjects with sarcoidosis disease. Phosphatidylethanolamine methyltransferase (PMT1) plays an important regulatory role in membrane signal transmission. cAMP and cGMP phosphodiesterases (PDE) regulate cytoplasmic cyclic nucleotide levels and so participate in the modulation of the cell cycle. We observed a decreased PMT1 activity in lymphocytes and monocytes and a decreased cAMP and cGMP PDE activities in monocytes. It is not now possible to say if these abnormalities are primary or secondary. Whatever the origin of this dysfunctioning, these results evoke simultaneous disturbances of membrane signal transmission and cell cycle in monocytes and membrane abnormalities in lymphocytes. These abnormalities could explain some immune cell defects in sarcoidosis disease.     

Inhibition of low Km cGMP phosphodiesterases and Ca+(+)-regulated protein kinases and relationship to vasorelaxation by cicletanine.

In the present studies we sought to determine if cicletanine, which is an antihypertensive agent of unknown mechanism, could alter cGMP metabolism via inhibition of cGMP phosphodiesterases (PDE) in vascular smooth muscle. Cicletanine was determined to be a mixed (competitive, noncompetitive) inhibitor of both calmodulin-regulated and cGMP-specific PDEs from monkey aortic smooth muscle with Ki values of 450 to 700 microM. Cicletanine also potentiated vasorelaxation by the guanylate cyclase activators sodium nitroprusside and atrial natriuretic peptide in isolated rat aortas. Potentiation was not dependent upon the contractile agonists nor was it indomethacin-sensitive. Neither potentiation nor inhibition of cGMP PDEs was stereoselective. Methylene blue attenuated a component of cicletanine-induced vasorelaxation, but did not completely obviate relaxation. Both cicletanine and the cGMP-PDE inhibitor zaprinast potentiated sodium nitroprusside-mediated cGMP formation and relaxation, although the increase in cGMP content was markedly greater with zaprinast compared to cicletanine. In further studies, cicletanine did not potentiate cGMP activation of cGMP-dependent protein kinase, but did inhibit calmodulin-activated myosin light chain kinase and protein kinase C at relatively high concentrations (approximately 1 mM). In summary, these data demonstrate that cicletanine inhibits vascular cGMP PDEs, potentiates vasorelaxation, and to a limited extent, cGMP formation by guanylate cyclase activators in vascular smooth muscle. However, these relationships for cicletanine are dissimilar from the reference cGMP PDE inhibitor, zaprinast. Thus, other mechanisms may also contribute to the vasorelaxant action of cicletanine.     

Nitric Oxide‐Induced Changes in Endothelial Expression of Phosphodiesterases 2, 3, and 5

(Headache 2010;50:431‐441) Objective.— To investigate nitric oxide (NO)‐mediated changes in expression of cyclic nucleotide degrading phosphodiesterases 2A (PDE2A), PDE3B, and PDE5A in human endothelial cells. Background.— Nitric oxide induces production of cyclic guanosine monophosphate (cGMP), which along with cyclic adenosine monophosphate (cAMP) is degraded by PDEs. NO donors and selective inhibitors of PDE3 and PDE5 induce migraine‐like headache and play a role in endothelial dysfunction during stroke. The current study investigates possible NO modulation of cGMP‐related PDEs relevant to headache induction in a cell line containing such PDEs. Methods.— Real time polymerase chain reaction and Western blots were used to show expression of PDE2A, PDE3B, and PDE5A in a stable cell line of human brain microvascular endothelial cells. Effects of NO on PDE expression were analyzed at specific time intervals after continued DETA NONOate administration. Results.— This study shows the expression of PDE2A, PDE3B, and PDE5A mRNA and PDE3B and PDE5A protein in human cerebral endothelial cells. Long‐term DETA NONOate administration induced an immediate mRNA up‐regulation of PDE5A (1.9‐fold, 0.5 hour), an early peak of PDE2A (1.4‐fold, 1 and 2 hours) and later up‐regulation of both PDE3B (1.6‐fold, 4 hours) and PDE2A (1.7‐fold, 8 hours and 1.2‐fold after 24 hours). Such changes were, however, not translated into significant changes in protein expression indicating few, if any, functional effects. Conclusions.— Long‐term NO stimulation modulated PDE3 and PDE5 mRNA expression in endothelial cells. However, PDE3 and PDE5 protein levels were unaffected by NO. The presence of PDE3 or PDE5 in endothelial cells indicates that selective inhibitors may have functional effects in such cells. A complex interaction of cGMP and cAMP in response to NO administration may take place if the mRNA translates into active protein. Whether or not this plays a role in the headache mechanisms remains to be investigated.     

Preclinical Characterization of the Phosphodiesterase 10A PET Tracer [<Superscript>11</Superscript>C]MK-8193

Purpose

A positron emission tomography (PET) tracer for the enzyme phosphodiesterase 10A (PDE10A) is desirable to guide the discovery and development of PDE10A inhibitors as potential therapeutics. The preclinical characterization of the PDE10A PET tracer [¹¹C]MK-8193 is described.

Procedures

In vitro binding studies with [³H]MK-8193 were conducted in rat, monkey, and human brain tissue. PET studies with [¹¹C]MK-8193 were conducted in rats and rhesus monkeys at baseline and following administration of a PDE10A inhibitor.

Results

[³H]MK-8193 is a high-affinity, selective PDE10A radioligand in rat, monkey, and human brain tissue. In vivo, [¹¹C]MK-8193 displays rapid kinetics, low test-retest variability, and a large specific signal that is displaced by a structurally diverse PDE10A inhibitor, enabling the determination of pharmacokinetic/enzyme occupancy relationships.

Conclusions

[¹¹C]MK-8193 is a useful PET tracer for the preclinical characterization of PDE10A therapeutic candidates in rat and monkey. Further evaluation of [¹¹C]MK-8193 in humans is warranted.

     

cGMP-stimulated cyclic nucleotide phosphodiesterase regulates the basal calcium current in human atrial myocytes.

EHNA (Erythro-9-[2-hydroxy-3-nonyl]adenine) is a wellknown inhibitor of adenosine deaminase. Recently, EHNA was shown to block the activity of purified soluble cGMPstimulated phosphodiesterase (PDE2) from frog, human, and porcine heart with an apparent Ki value of approximately 1 microM and with negligible effects on Ca2+/calmodulin PDE (PDE1), cGMP-inhibited PDE (PDE3), and low Km cAMP-specific PDE (PDE4) (Méry, P.F., C. Pavoine, F. Pecker, and R. Fischmeister. 1995. Mol. Pharmacol. 48:121-130; Podzuweit, T., P. Nennstiel, and A. Muller. 1995. Cell. Signalling. 7:733- 738). To investigate the role of PDE2 in the regulation of cardiac L-type Ca2+ current (ICa), we have examined the effect of EHNA on ICa in freshly isolated human atrial myocytes. Extracellular application of 0.1-10 microM EHNA induced an increase in the amplitude of basal ICa ( approximately 80% at 1 microM) without modification of the current-voltage or inactivation curves. The maximal stimulatory effect of EHNA on ICa was comparable in amplitude with the maximal effect of isoprenaline (1 microM), and the two effects were not additive. The effect of EHNA was not a result of adenosine deaminase inhibition, since 2'-deoxycoformycin (1-30 microM), another adenosine deaminase inhibitor with no effect on PDE2, or adenosine (1-10 microM) did not increase ICa. In the absence of intracellular GTP, the substrate of guanylyl cyclase, EHNA did not increase ICa. However, under similar conditions, intracellular perfusion with 0.5 microM cGMP produced an 80% increase in ICa. As opposed to human cardiomyocytes, EHNA (1-10 microM) did not modify ICa in isolated rat ventricular and atrial myocytes. We conclude that basal ICa is controlled by PDE2 activity in human atrial myocytes. Both PDE2 and PDE3 may contribute to keep the cyclic nucleotides concentrations at minimum in the absence of adenylyl and/or guanylyl cyclase stimulation.     

Physostigmine inhibition of 3',5'-cyclic AMP phosphodiesterase from cat sciatic nerve

This study was designed to determine whether cholinergic drug interaction with cyclic (c) AMP phosphodiesterase (PDE) might account for part of the effects of this class of drugs at the neuromuscular junction. The activity levels of both high- and low-affinity forms of cAMP PDE from cat sciatic nerve were examined for drug inhibition or activation. Of the cholinergic drugs tested, only physostigmine and Tacrine produced significant cAMP PDE inhibition. Physostigmine was 10 times more potent than theophylline and half as potent as SQ 20,009 (known PDE inhibitors) in inhibiting motor nerve cAMP PDE. Tacrine inhibited this enzyme at concentrations comparable with theophylline. None of the other drugs tested (diisopropylfluorophosphate, edrophonium, neostigmine, ecothiophate, carbachol or d-tubocurarine) produced significant changes in cAMP PDE activity. The inhibitory effects of physostigmine were shown to be pH independent over a range of 7.0 to 8.5. Kinetic studies indicated a mixed form of inhibition for physostigmine and Tacrine comparable with that seen for theophylline. These data indicate that the anticholinesterase activity of physostigmine and Tacrine do not adequately describe the facilitatory actions of these drugs at the motor nerve ending.     

Leptin inhibits insulin secretion by activation of phosphodiesterase 3B.

The molecular signaling events by which leptin exerts its functions in vivo are not well delineated. Here, we show a novel leptin signaling mechanism that requires phosphoinositide 3-kinase (PI 3-kinase)-dependent activation of cyclic nucleotide phosphodiesterase 3B (PDE3B) and subsequent suppression of cAMP levels. In pancreatic beta cells, leptin causes the activation of PDE3B, which leads to marked inhibition of glucagon-like peptide-1-stimulated insulin secretion. The effect of leptin is abolished when insulin secretion is induced with cAMP analogues that cannot be hydrolyzed by PDE3B. Selective inhibitors of PDE3B and PI 3-kinase completely prevent the leptin effect on insulin secretion and cAMP accumulation. The results demonstrate that one of the physiological effects of leptin, suppression of insulin secretion, is mediated through activation of PDE3B and suggest PDE3B as a mediator of leptin action in other tissues.     

The role of phosphodiesterase in mediating the effect of protein kinase C on cyclic AMP accumulation upon kappa-opioid receptor stimulation in the rat heart

This study determined whether phosphodiesterase (PDE) was activated by protein kinase C (PKC) upon kappa-receptor stimulation, and if so, to identify the isozyme. We first studied the effects of trans-(+/-)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl] cyclohexyl) benzeneacetamide methanesulphonate (U50,488H), a selective kappa-opioid receptor (OR) agonist, and phorbol-12-myristate-13-acetate (PMA), a PKC activator, on cAMP accumulation and PDE activity in rat ventricular myocytes when PKC and PDE were inhibited by respective inhibitors. Like PMA, U50,488H decreased the forskolin-stimulated cAMP accumulation and dose-dependently stimulated the PDE activity, which were antagonized by 10(-6) M chelerythrine and bisindolylmaleimide I, selective PKC antagonists. In addition, 3-isobutyl-1-methylxanthine, a PDE inhibitor, dose-dependently attenuated the inhibition on forskolin-stimulated cAMP accumulation and abolished the stimulation on PDE activity by U50,488H and PMA. The observations suggest that PKC may enhance cAMP degradation through activating PDE upon kappa-OR stimulation. To identify the isozyme(s) mediating the effect of PKC upon kappa-OR stimulation, selective inhibitors were used. We found that 10(-5) M Ro-20-1724, a selective cAMP-specific PDE (PDE-IV) inhibitor, abolished the inhibitory effects of U50,488H and PMA, whereas 8-methoxymethyl-3-isobutyl-1-methylxanthine, erythro-9-(2-hydroxy-3-nonyl) adenine, cilostamide, and zaprinast, selective inhibitors of Ca(2+)/calmodulin-dependent PDE (PDE-I), cGMP-stimulated PDE (PDE-II), cGMP-inhibited PDE (PDE-III), and cGMP-specific PDE (PDE-V), respectively, had no effect. Moreover, rolipram, another selective PDE-IV inhibitor, also dose-dependently attenuated the inhibition on forskolin-stimulated cAMP accumulation and stimulation on PDE activity by U50,488H and PMA. In conclusion, this study has provided evidence for the first time that PKC and PDE-IV mediate the action of kappa-OR.