Calmodulin-dependent Phosphodiesterase Research Articles

Airway Ciliary Beating Affected by the Pcp4 Dose-Dependent [Ca2+]i Increase in Down Syndrome Mice, Ts1Rhr

In Ts1Rhr, a Down syndrome model mouse, the airway ciliary beatings are impaired; that is, decreases in ciliary beat frequency (CBF) and ciliary bend angle (CBA, an index of ciliary beat amplitude)). A resumption to two copies of the Pcp4 gene on the Ts1Rhr trisomic segment (Ts1Rhr:Pcp4+/+/-) rescues the decreases in CBF and CBA that occur in Ts1Rhr. In airway cilia, upon stimulation with procaterol (a β2-agonist), the CBF increase is slower over the time course than the CBA increase because of cAMP degradation by Ca2+/calmodulin-dependent phosphodiesterase 1 (PDE1) existing in the metabolon regulating CBF. In Ts1Rhr, procaterol-stimulated CBF increase was much slower over the time course than in the wild-type mouse (Wt) or Ts1Rhr:Pcp4+/+/-. However, in the presence of 8MmIBMX (8-methoxymethyl isobutylmethyl xanthine, an inhibitor of PDE1) or calmidazolium (an inhibitor of calmodulin), in both Wt and Ts1Rhr, procaterol stimulates CBF and CBA increases over a similar time course. Measurements of cAMP revealed that the cAMP contents were lower in Ts1Rhr than in Wt or in Ts1Rhr:Pcp4+/+/-, suggesting the activation of PDE1A that is present in Ts1Rhr airway cilia. Measurements of the intracellular Ca2+ concentration ([Ca2+]i) in airway ciliary cells revealed that temperature (increasing from 25 to 37 °C) or 4αPDD (a selective transient receptor potential vanilloid 4 (TRPV4) agonist) stimulates a larger [Ca2+]i increase in Ts1Rhr than in Wt or Ts1Rhr:Pcp4+/+/-. In airway ciliary cells of Ts1Rhr, Pcp4-dose dependent activation of TRPV4 appears to induce an increase in the basal [Ca2+]i. In early embryonic day mice, a basal [Ca2+]i increased by PCP4 expressed may affect axonemal regulatory complexes regulated by the Ca2+-signal in Ts1Rhr, leading to a decrease in the basal CBF and CBA of airway cilia.

Pharmacological investigation of biochemical and behavioral effects resembling Huntington's disease utilizing selective calcium and calmodulin dependent phosphodiesterase inhibitor

Pharmacological investigation of biochemical and behavioral effects resembling Huntington's disease utilizing selective calcium and calmodulin dependent phosphodiesterase inhibitor

Transport, Bioavailability, Safety, and Calmodulin-Dependent-Phosphodiesterase-Inhibitory Properties of Flaxseed-Derived Bioactive Peptides

The aim of this work was to determine bioavailability and in vivo calmodulin-dependent-phosphodiesterase (CaMPDE)-inhibitory activity of six flaxseed-protein-derived peptides (AGA, AKLMS, QIAK, RWIQ, QQAKQ, and KQLSTGC) after oral administration to Wistar rats. Initial experiments tested the cytotoxicity and cellular-transport potentials of the peptides using Caco-2 cells. The cytotoxicity assay indicated that none of the six peptides had an adverse effect on the proliferation and viability of the Caco-2 cells, whereas the transport assay confirmed peptide translocation across the cell membrane. However, only two of the peptides (AGA and RWIQ) were detected in the rat serum up to 90 min postgavage, with traces of RWIQ persisting in serum 1 week after oral gavage. The six peptides inhibited plasma activity of CaMPDE with AGA (34.63%), QIAK (36.66%), and KQLSTGC (34.21%) being the most effective 30 min after gavage. In contrast, only AGA maintained significant plasma-CaMPDE-activity inhibition (44.35%) after 60 min.

Pleiotropic roles of Ca+2/calmodulin-dependent pathways in regulating cadmium-induced toxicity in human osteoblast-like cell lines

The heavy metal cadmium is a widespread environmental contaminant that has gained public attention due to the global increase in cadmium-containing electronic waste. Human exposure to cadmium is linked to the pathogenesis of osteoporosis. We previously reported cadmium induces apoptosis and decreases alkaline phosphatase mRNA expression via extracellular signal-regulated protein kinase (ERK) activation in Saos-2 bone-forming osteoblasts. This study examines the mechanisms of cadmium-induced osteotoxicity by investigating roles of Ca+2/calmodulin-dependent protein kinase (CAMK) pathways. Saos-2 or MG-63 cells were treated for 24 or 48h with 5μM CdCl2 alone or in combination with calmodulin-dependent phosphodiesterase (PDE) inhibitor CGS-9343β; calmodulin-dependent kinase kinase (CAMKK) inhibitor STO-609; or calmodulin-dependent kinase II (CAMKII) inhibitor KN-93. CGS-9343β protected against cadmium-induced toxicity and attenuated ERK activation; STO-609 enhanced toxicity and exacerbated ERK activation, whereas KN-93 had no detectable effect on cadmium-induced toxicity. Furthermore, CGS-9343β co-treatment attenuated cadmium-induced apoptosis; but CGS-9343β did not recover cadmium-induced decrease in ALP activity. The major findings suggest the calmodulin-dependent PDE pathway facilitates cadmium-induced ERK activation leading to apoptosis, whereas the CAMKK pathway plays a protective role against cadmium-induced osteotoxicity via ERK signaling. This research distinguishes itself by identifying pleiotropic roles for CAMK pathways in mediating cadmium’s toxicity in osteoblasts.

Modulation of Polycystic Kidney Disease Severity by Phosphodiesterase 1 and 3 Subfamilies.

Aberrant intracellular calcium levels and increased cAMP signaling contribute to the development of polycystic kidney disease (PKD). cAMP can be hydrolyzed by various phosphodiesterases (PDEs). To examine the role of cAMP hydrolysis and the most relevant PDEs in the pathogenesis of PKD, we examined cyst development in Pde1- or Pde3-knockout mice on the Pkd2(-/WS25) background (WS25 is an unstable Pkd2 allele). These PDEs were selected because of their importance in cross-talk between calcium and cyclic nucleotide signaling (PDE1), control of cell proliferation and cystic fibrosis transmembrane conductance regulator (CFTR) -driven fluid secretion (PDE3), and response to vasopressin V2 receptor activation (both). In Pkd2(-/WS25) mice, knockout of Pde1a, Pde1c, or Pde3a but not of Pde1b or Pde3b aggravated the development of PKD and was associated with higher levels of protein kinase A-phosphorylated (Ser133) cAMP-responsive binding protein (P-CREB), activating transcription factor-1, and CREB-induced CRE modulator proteins in kidney nuclear preparations. Immunostaining also revealed higher expression of P-CREB in Pkd2(-/) (WS25);Pde1a(-/-), Pkd2(-) (/WS25);Pde1c(-/-), and Pkd2(-/) (WS25);Pde3a(-/-) kidneys. The cystogenic effect of desmopressin administration was markedly enhanced in Pkd2(-/WS25);Pde3a(-/-) mice, despite PDE3 accounting for only a small fraction of renal cAMP PDE activity. These observations show that calcium- and calmodulin-dependent PDEs (PDE1A and PDE1C) and PDE3A modulate the development of PKD, possibly through the regulation of compartmentalized cAMP pools that control cell proliferation and CFTR-driven fluid secretion. Treatments capable of increasing the expression or activity of these PDEs may, therefore, retard the development of PKD.

Endothelium-independent and endothelium-dependent vasorelaxation by a dichloromethane fraction from <i>Anogeissus leiocarpus</i> (dc) <i>guill</i>. Et <i>Perr. (combretaceae)</i>: possible involvement of cyclic nucleotide phosphodiesterase inhibition

Many traditional medicinal herbs from Burkina Faso are used to treat arterial hypertension (HTA). Among them, Anogeissus leiocarpus (A. Leiocarpus) which is well known and widely used in Burkina traditional medicine. Herein we assess the effects of dichloromethane fraction from A. leiocarpus stem bark (ALF), selected as the most active on cyclic nucleotide phosphodiesterases (PDEs) and characterized its specificity towards purified vascular PDE1 to PDE5 isoenzymes and study its effects on a vascular model. ALF potently and preferentially inhibits (IC50=1.6 ± 0.6 µg/mL) the calmodulin-dependent phosphodiesterase PDE1, being mainly present in vascular smooth muscle and preferentially hydrolyses cGMP. In the same range (IC50 =2.8 ± 0.2 µg/ml) ALF inhibits PDE2, a cGMP-activated enzyme that is only present in endothelial cells and hydrolyses both cAMP and cGMP. PDE5, which specifically hydrolyses cGMP and which mainly contributes to cGMP hydrolysis is also potently inhibited by ALF (IC50=7.6 ± 3.5 µg/ml). The potencies of ALF on cAMP hydrolyzing isoenzymes was lesser, being more effective on PDE4 (IC50= 17.6 ± 3.5 µg/ml) than on PDE3 (60.9 ± 1.8 µg/ml). Since the major effect of ALF were against cGMP hydrolysis and since cGMP is implicated in endothelium-dependent relaxation, the endothelium-dependent vasorelaxation was studied on isolated porcine coronary arteries rings pre-contracted with U46619. The endothelium-dependent vasorelaxation is significantly inhibited by N(ω)-nitro-L-arginine (LNA 300 µmol/L, an inhibitor of endothelial NO synthase), but not affected by charybdotoxin (CTX, 100 nM) plus apamin (APA, 100 nM) (two inhibitors of EDHF-mediated responses). The combination of 4-aminopyridine (4-AP, 1 mmol/L, inhibitor of voltage-dependent potassium channels, Kv) plus baryum (Ba(2+), 30 µmol/L, inhibitor of the potassium channels with entering correction, Kir) plus ouabain (3 µmol/L, inhibitor of ATPase Na(+)/K(+) channels) partially inhibits endothelium-independent vasorelaxant effect. This endothelium-independent relaxant effect was also sensitive to combination of 1H-[1,2,4]-oxadiazole-[4,3-α]-quinoxalin1-one (ODQ, 10 µM, soluble guanylyl cyclase inhibitor) and N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide dihydrochloride (H89, 100 nM, Protein Kinase A inhibitor). Taken together, these results indicate that ALF is a powerful vasodilator modulated by the formation of NO from endothelium, but also act by directly relaxing the vascular smooth muscle cells, by inhibiting cGMP hydrolyzing PDEs (PDE1, PDE2 and PDE5) and to a lesser extend on cAMP degradation (PDE3 and PDE4), cAMP and cGMP being second messengers involved in vascular relaxation.

Possible Involvement of Calmodulin in the Spasmolytic Effect of Extracts from Sabal serrulata Fruits

The effect of total lipidic and saponifiable extracts from Sabal serrulata fruits on contraction induced by vanadate (0–3 mM) in the uterus of Wistar rats incubated in calcium-free solution have been assayed. The two extracts relaxed the contraction induced by vanadate. The EC50 values were 43.9 ± 8.4 and 11.4 ± 1.4 μg mL−1 for total lipid and saponifiable material, respectively. Total lipid and saponifiable extracts also inhibited calmodulin-dependent cAMP phosphodiesterase activity in a dose-dependent way. The EC50 values were 25.1 ± 4 and 28.6 ± 4 μg mL−1 for total lipid and saponifiable material, respectively. Phosphodiesterase basal activity was only modified with the maximum concentration (100 μg mL−1). These results suggest that the effects of extracts from Sabal serrulata fruits could be due, at least partially, to the inhibition of calmodulin but other mechanisms cannot be excluded.

Identification and inhibitory properties of multifunctional peptides from pea protein hydrolysate.

Pea protein isolate was hydrolyzed with alcalase, and the hydrolysate passed through a 1 kDa cutoff ultrafiltration membrane. The permeate was freeze-dried and fractionated on a cationic solid-phase extraction (SPE) column. All fractions were tested for their inhibitory activities against angiotensin-converting enzyme (ACE), renin, and calmodulin-dependent phosphodiesterase 1 (CaMPDE). With the exception of the first eluted fraction, inhibitory properties of the SPE fractions against CaMPDE (but not ACE and renin) were directly related to cationic character (residence time on the column). However, the fraction that eluted with 1% ammonium hydroxide (SPE 1%) had the highest peptide yield and was subsequently fractionated using two consecutive rounds of reversed-phase high-performance liquid chromatography to obtain three peaks with major peptides identified as IR, KF, and EF by ultra performance liquid chromatography-tandem mass spectrometry. The three dipeptides showed weak inhibitory properties toward CaMPDE but strong inhibitions (IC50 values <25 mM) of ACE and renin. In general, the peptides had higher potency against ACE than against renin. It is indicated from our results that these peptides may be used as potential ingredients to formulate multifunctional food products and nutraceuticals.

Cyclic nucleotide signaling in polycystic kidney disease

Increased levels of 3'-5'-cyclic adenosine monophosphate (cAMP) stimulate cell proliferation and fluid secretion in polycystic kidney disease. Levels of this molecule are more sensitive to inhibition of phosphodiesterases (PDEs), whose activity far exceeds the rate of cAMP synthesis by adenylyl cyclase. Several PDEs exist, and here we measured the activity and expression of PDE families, their isoforms, and the expression of downstream effectors of cAMP signaling in the kidneys of rodents with polycystic kidney disease. We found a higher overall PDE activity in kidneys from mice as compared with rats, as well as a higher contribution of PDE1, relative to PDE4 and PDE3, to total PDE activity of kidney lysates and lower PDE1, PDE3, and PDE4 activities in the kidneys of cystic as compared with wild-type mice. There were reduced amounts of several PDE1, PDE3, and PDE4 proteins, possibly due to increased protein degradation despite an upregulation of their mRNA. Increased levels of cGMP were found in the kidneys of cystic animals, suggesting in vivo downregulation of PDE1 activity. We found an additive stimulatory effect of cAMP and cGMP on cystogenesis in vitro. Cyclic AMP-dependent protein kinase subunits Ialpha and IIbeta, PKare, the transcription factor CREB-1 mRNA, and CREM, ATF-1, and ICER proteins were upregulated in the kidneys of cystic as compared with wild-type animals. Our study suggests that alterations in cyclic nucleotide catabolism may render cystic epithelium particularly susceptible to factors acting on Gs-coupled receptors. This may account, in part, for increased cyclic nucleotide signaling in polycystic kidney disease and contribute substantially to disease progression.

Multifunctional peptides from egg white lysozyme

Abstract Hen’s egg white lysozyme (HEWL) is one of the major egg white proteins with well demonstrated antimicrobial activity. Bioactive peptides other than antimicrobial peptides from HEWL have not been reported; therefore, the purpose of the study was to explore new bioactivities of lysozyme-derived bioactive peptides. HEWL was hydrolysed with Alcalase and fractionated by cation-exchange chromatography. The Alcalase HEWL hydrolysate and its fractions were analyzed for inhibitory activities against calmodulin-dependent phosphodiesterase (CaMPDE) and antioxidant activities using oxygen radical absorbance capacity-fluorescein (ORAC-FL), 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radical cation (ABTS + ) and 2,2-di(4- tert -octylphenyl)-1-picrylhydrazyl (DPPH) radical scavenging methods. The fractionated peptides had higher CaMPDE inhibition activity, ORAC-FL value and ABTS + scavenging activity than those of the hydrolysate. Peptide sequences in the most overall active fractions were characterized by LC–MS/MS. Our results showed that HEWL hydrolysate and its peptide fractions may serve as useful ingredients in the formulation of functional foods and nutraceuticals.

Dihydroartemisinin can inhibit calmodulin, calmodulin-dependent phosphodiesterase activity and stimulate cellular immune responses

Calmodulin (CaM) is a ubiquitous, calcium-binding protein that regulates several important aspects of cellular metabolism. A number of enzymes such as phosphodiesterase (PDE-1) are stimulated by CaM. In previous studies, our results showed that artemisinin (ART) is a potent inhibitor of CaM and PDE-1 activity. In this study, the effects of dihydroartemisinin (DHA) that is a semisynthesized agent from the ART on CaM structure were investigated. The result showed that DHA increased fluorescence emission of CaM in higher amounts compared with the ART. Also, the effect of DHA on CaM-dependent PDE-1 activity was studied. Kinetic analysis of the DHA–CaM interaction showed that this agent competitively inhibited the activation of PDE-1 without affecting Vmax. Km values of PDE-1 in the presence of ART and DHA were 10 and 15 µM, respectively; DHA increased Km value in higher amounts compared with the ART. The Ki constants for ART and DHA were 10 µM and 7.3 µM, respectively. As a conclusion, CaM and CaM-dependent PDE-1 were inhibited by DHA more than ART. The data indicated that DHA could stimulate the delayed type hypersensitivity (DTH) against sheep blood cells in Balb/c mice and reduced the tumor growth in vivo against invasive ductal carcinoma in Balb/c mice.

Calmodulin inhibitory activity of the malbrancheamides and various analogs

The preparation and biological activity of various structural analogs of the malbrancheamides are disclosed. The impact of indole chlorination, C-12a relative stereochemistry, and bicyclo[2.2.2]diazaoctane core oxidation state on the ability of these analogs to inhibit calmodulin dependent phosphodiesterase (PDE1) was studied, and a number of potent compounds were identified. ▪

Gene expression and protein localization of calmodulin‐dependent phosphodiesterase during ontogenesis of chick retina

Calmodulin-dependent phosphodiesterase (PDE1) is a key enzyme in cyclic nucleotides metabolism. We studied its gene expression and protein localization during retinal development in chick embryos. Western blot and densitometric analysis demonstrated that the expression of the three isoforms changed during development. PDE1A was highly expressed at the early stages and decreased as development proceeded. PDE1B expression remained relatively low and constant over time. PDE1C showed a prominent increase (13-fold) between embryonic day (E) 7 and E13, followed by a moderate increase between E13 and postnatal day (P) 1. The presence of the enzyme in the different retinal locations was strongly modulated by development. PDE1A immunostaining was first detected at the ganglion cell level (E7), then in the outer retina (E15-E21). At P5, the immunostaining was confined in the optic fiber layer. Isoform C immunolocalization followed the same inner-outer pattern as isoform A. At 5 days posthatching (P5), the immunoreactivity was restricted, as well as for the isoform A, in the optic fiber layer. The isoform B immunolabelling was low and evenly distributed across the retina at all stages. The different developmental profiles of PDE1A, PDE1B, and PDE1C induced a temporal modulation in cyclic nucleotides concentration, suggesting specific roles of this enzyme in the morphofunctional development of retinal circuitry.

Kinetic properties of Ca 2+/calmodulin-dependent phosphodiesterase isoforms dictate intracellular cAMP dynamics in response to elevation of cytosolic Ca 2+

Multiply regulated adenylyl cyclases (AC) and phosphodiesterases (PDE) can yield complex intracellular cAMP signals. Ca 2+-sensitive ACs have received far greater attention than the Ca 2+/calmodulin-dependent PDE (PDE1) family in governing intracellular cAMP dynamics in response to changes in the cytosolic Ca 2+ concentration ([Ca 2+] i ). Here, we have stably expressed two isoforms of PDE1, PDE1A2 and PDE1C4, in HEK-293 cells to determine whether they exert different impacts on cellular cAMP. Fractionation and imaging showed that both PDEs occurred mainly in the cytosol. However, PDE1A2 and PDE1C4 differed considerably in their ability to hydrolyze cAMP and in their susceptibility to inhibition by the non-selective PDE inhibitor, IBMX and the PDE1-selective inhibitor, MMX. PDE1A2 had an approximately 30-fold greater K m for cAMP than PDE1C4 and yet was more susceptible to inhibition by IBMX and MMX than was PDE1C4. These differences were mirrored in intact cells when thapsigargin-induced capacitative Ca 2+ entry (CCE) activated the PDEs. Mirroring their kinetic properties, PDE1C4 was active at near basal cAMP levels, whereas PDE1A2 required agonist-triggered levels of cAMP, produced in response to stimulation of ACs. The effectiveness of IBMX and MMX to inhibit PDE1A2 and PDE1C4 in functional studies was inversely related to their respective affinities for cAMP. To assess the impact of the two isoforms on cAMP dynamics, real-time cAMP measurements were performed in single cells expressing the two PDE isoforms and a fluorescent Epac-1 cAMP biosensor, in response to CCE. These measurements showed that prostaglandin E 1-mediated cAMP production was markedly attenuated in PDE1C4-expressing cells upon induction of CCE and cAMP hydrolysis occurred at a faster rate than in cells expressing PDE1A2 under similar conditions. These results prove that the kinetic properties of PDE isoforms play a major role in determining intracellular cAMP signals in response to physiological elevation of [Ca 2+] i and thereby provide a rationale for the utility of diverse PDE1 species.

Structure-activity relationships of dimethylsphingosine (DMS) derivatives and their effects on intracellular pH and Ca2+ in the U937 monocyte cell line

We recently reported that dimethylsphingosine (DMS), a metabolite of sphingolipids, increased intracellular pH and Ca2+ concentration in U937 human monocytes. In the present study, we found that dimethylphytosphingosine (DMPH) induced the above responses more robustly than DMS. However, phytosphingosine, monomethylphytosphingosine or trimethylsphingosine showed little or no activity. Synthetic C3 deoxy analogues of sphingosine did show similar activities, with the C16 analogue more so than C18. The following structure-activity relationships were observed between DMS derivatives and the intracellular pH and Ca2+ concentrations in U937 monocytes; 1) dimethyl modification is important for the DMS-induced increase of intracellular pH and Ca2+, 2) the addition of an OH group on C4 enhances both activities, 3) the deletion of the OH group on C3 has a negligible effect on the activities, and 4) C16 appears to be more effective than C18. We also found that W-7, a calmodulin inhibitor, blocked the DMS-induced pH increase, whereas, KN-62, ML9, and MMPX, specific inhibitors for calmodulin-dependent kinase II, myosin light chain kinase, and Ca(2+)-calmodulin-dependent phosphodiesterase, respectively, did not affect DMS-induced increases of pH in the U937 monocytes.

Eburnamine Derivatives and the Brain

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Potential role of calmodulin-dependent phosphodiesterase in human brain tumor (Review)

A wealth of knowledge exists regarding calmodulin-dependent phosphodiesterase (PDE1) in normal mammalian tissues. However, much remains to be known about the enzyme in human brain tumors (glioblastoma multiforme, GBM). PDE1 has not been investigated in GBM relative to normal brain and remains an important area of investigation. In this review, we have summarized the works that have been carried out in our laboratory on the potential role of PDE1 in GBM. In the normal brain, PDE1 is localized in all four principal parts of the brain: cerebrum, diencephalon, brainstem and cerebellum. Specifically, PDE1 is concentrated in anatomically distinct regions of the brain including the striatum, globus pallidus, substantia nigra, subiculum, Purkinjie cells, the external layer of the developing cerebellum and the cerebral cortex. In contrast, PDE1 is found to be very low in the GBM tissue compared to normal cerebral cortex tissue. However, the discovery of an inhibitor in the tumor tissue is interesting and definitely warrants further investigation, particularly, in relation to its possible interaction with PDE1.

Eburnamine derivatives and the brain

The Apocynaceae plant family contains a great number of so called eburnamine-vincamine alkaloids. Quite a few of these alkaloids exert varied pharmacological activities on the cell multiplication, cardiovascular system, and brain functions. Many derivatives were also synthesized to find pharmacologically active compounds better characterized and safer to be administered than the natural plant alkaloids themselves. We concentrate on the eburnamine structures with cerebral activities in this review. Vincamine, vinburnine, vindeburnol, apovincaminate, and vinpocetine (cis-ethyl-apovincaminate) all share modulatory effects on brain circulation and neuronal homeostasis, bear antihypoxic and neuroprotective potencies to various degrees. The most eminent compound of this class of alkaloids is vinpocetine. Since its introduction to the market as a neuroprotective agent many non clinical and clinical studies proved vinpocetine's effects on calmodulin dependent phosphodiesterase E1, on sodium, calcium channels, peripheral benzodiazepine receptor, and glutamate receptors as well as its clinical usefulness in the treatment of post-ischaemic stroke disease states and various disorders of cerebrovascular origin. Lately, positron emission tomography studies proved that vinpocetine has a rapid uptake in the primate and human brain with a heterogeneous distribution pattern (preference areas: thalamus, basal ganglia, and visual cortex) both after intravenous and oral administration. Vinpocetine exerts beneficial effects in cerebral glucose metabolism and regional cerebral blood flow in chronic post-stroke patients.

Sustained Entry of Ca2+ Is Required to Activate Ca2+-Calmodulin-dependent Phosphodiesterase 1A

Regulation of adenylyl cyclases (ACs) by Ca2+ requires capacitative Ca2+ entry (CCE) (Cooper, D. M. F. (2003) Biochem. J. 375, 517-529), but whether Ca2+-sensitive phosphodiesterases (PDEs) are similarly discriminating has never been addressed. In the present study, a variety of conditions were devised to manipulate [Ca2+]i so that we could ask whether PDE1 selectively responds to different modes of elevating [Ca2+]i, viz. Ca2+ released from intracellular stores and various modes of Ca2+ entry. In 1321N1 human astrocytoma cells, the endogenous PDE1 (identified as PDE1A by reverse transcriptase-PCR) was largely insensitive to Ca2+ released from carbachol-sensitive stores but was robustly stimulated by a similar rise in [Ca2+]i due to carbachol-induced Ca2+ influx. Gd3+, which effectively blocked thapsigargin-induced CCE and its effect on PDE1A, also inhibited the activation of PDE1A by carbachol-induced Ca2+ entry. However, non-selective ionomycin-mediated Ca2+ entry also activated PDE1A, so that, unlike Ca2+-sensitive ACs, PDE1A cannot discriminate between the different sources of Ca2+ entry. Fractionation of the cells revealed that the Ca2+-calmodulin-stimulated PDE activity was not present at the plasma membrane but was associated with the cytosol and the organellar compartments of the cell. Therefore, the apparent disparity between PDE1A and ACs is likely to be the consequence of their differential subcellular localization. Nevertheless, in a physiological context, where artificial modes of elevating [Ca2+]i are not available, as with ACs, a dependence on CCE would be evident, and it would be the duration of this influx of Ca2+ that would determine how long PDE1A was activated.

Cyclic GMP phosphodiesterases and regulation of smooth muscle function.

Cyclic GMP (cGMP) made in response to atrial natriuretic peptide (ANP) or nitric oxide (NO) is an important regulator of short-term changes in smooth muscle tone and longer-term responses to chronic drug treatment or proliferative signals. The ability of smooth muscle cells (SMCs) to utilize different combinations of phosphodiesterase (PDE) isozymes allows cGMP to mediate these multiple processes. For example, PDE5 as a major cGMP-hydrolyzing PDE effectively controls the development of smooth muscle relaxation. In order for contraction to occur, PDE5 is activated and cGMP falls. Conversely, blockade of PDE5 activity allows the relaxation cycle to be prolonged and enhanced. A recently shown direct activation of PDE5 by cGMP binding to the GAF A domain suggests that this regulatory site might be a target for new drug development. The calcium surge associated with vasoconstrictor initiated contraction also activates a calcium/calmodulin-dependent PDE (PDE1A). Together, PDE5 and PDE1A lower cGMP sufficiently to allow contraction. Longer term, both PDE5 and PDE1A mRNA are induced by chronic stimulation of guanylyl cyclase. This induction is a major cause of the tolerance that develops to NO-releasing drugs. Finally, high levels of cGMP or cAMP also act as a brake to attenuate the proliferative response of SMCs to many mitogens. After vessel damage, in order for SMC proliferation to occur, the levels of cGMP and cAMP must be decreased. In humans, this decrease is caused in large part by induction of another Ca2+/calmodulin-dependent PDE (PDE1C) that allows the brake to be released and proliferation to start.