Endogenous or overexpressed cGMP-dependent protein kinases inhibit cAMP-dependent renin release from rat isolated perfused kidney, microdissected glomeruli, and isolated juxtaglomerular cells

An overactive renin-angiotensin-aldosterone system (RAAS) has a central role in the pathogenesis of hypertension and cardiac hypertrophy, precursors of cardiac failure. Natriuretic peptides and NO acting through their second messenger, cGMP, increase natriuresis and diuresis, and inhibit renin release; however the mechanism by which this inhibition of the RAAS system functions is obscure. We recently reported cloning of the cDNA for type II cGMP-dependent protein kinase (cGK II), elucidated its first known function of inhibiting the cystic fibrosis transmembrane conductance regulator in rat intestine, and initially described its location in rat kidney juxtaglomerular (JG) cells, the ascending thin limb, and the brush border of proximal tubules. Here, we demonstrate inhibition of isoproterenol- or forskolin-stimulated renin release by 8-para-chlorophenylthio-cGMP (8-pCPT-cGMP), a selective activator of cGK, and prevention of this inhibition by a selective inhibitor of cGK, Rp-8-pCPT-cGMPS. In systems of differing complexity, inhibition by 8-pCPT-cGMP was nearly complete in isolated perfused kidney and microdissected afferent arterioles but only ≈25% in isolated JG cells. Expression of either cGK II or cGK I in JG cells by using adenoviral vectors enhanced the inhibition of forskolin-stimulated renin release by 8-pCPT-cGMP to 50%. Our results indicate that cGK II, and possibly cGK I, can mediate cGMP inhibitory effects on renin release and are physiological components of the cGMP signal transduction system which opposes the RAAS.     

Cellular distribution of phosphodiesterase isoforms in rat cardiac tissue.

We have resolved multiple forms of cyclic nucleotide phosphodiesterase (PDE) in whole rat ventricle and in isolated rat ventricular myocytes by use of anion-exchange high-performance liquid chromatography. One major form, the soluble calmodulin-stimulated PDE, is apparently absent from isolated myocytes. We discern four peaks of PDE activity (designated A-D in the order of their elution) in a soluble fraction obtained from whole rat ventricle. Peak A is stimulated twofold to threefold by the addition of calcium and calmodulin (Ca2+/CalM) and preferentially hydrolyzes cGMP over cAMP (in the presence of Ca2+/CalM, KmcGMP = 1.5 microM, KmcAMP = 17 microM). Peak B has similar affinities for both cAMP and cGMP (half-maximum velocities achieved at 30 microM substrate) and demonstrates positive cooperativity with cAMP but not with cGMP. The hydrolysis of cAMP by peak B is stimulated by cGMP at substrate concentrations up to 20 microM; the maximum effect is seen at 1 microM cAMP (25-fold stimulation by 3 microM cGMP). This pattern of stimulation by cGMP results from two kinetic changes: an increase in the enzyme's apparent affinity for cAMP (apparent KmcAMP decreases from 33 to 11 microM) and the abolition of positive cooperativity. Peaks C and D selectively hydrolyze cAMP, are not stimulated by Ca2+/CalM or cGMP, and differ in their affinities for substrate (peak C, apparent KmcAMP = 7.2 microM; peak D, 0.44 microM). In addition, peak D is much more sensitive than peak C to inhibition by cGMP, cilostamide, rolipram, and milrinone. Ro20-1724 is a slightly more potent inhibitor of peak D than of peak C. Peak D appears to consist of two different enzyme activities, one inhibited by cGMP, cilostamide, and cardiotonic drugs and the other potently inhibited by rolipram. In contrast to whole ventricle, the soluble fraction of isolated rat ventricular myocytes lacks peak A. Three major peaks in myocytes are entirely analogous to peaks B, C, and D of whole ventricle in terms of the NaCl concentration at which they elute, substrate affinities, and stimulation or inhibition by various drugs and effectors. We conclude that the soluble Ca2+/CalM-stimulated PDE in whole rat ventricle is present in nonmyocyte cells.     

Role of cGMP-inhibited phosphodiesterase and sarcoplasmic calcium in mediating the increase in basal heart rate with nitric oxide donors

Nitric oxide (NO) donors increase heart rate (HR) through a guanylyl cyclase-dependent stimulation of the pacemaker current I(f), without affecting basal I(Ca-L). The activity of I(f)is known to be enhanced by cyclic nucleotides and by an increase in cytosolic Ca(2+). We examined the role of cGMP-dependent signaling pathways and intracellular Ca(2+)stores in mediating the positive chronotropic effect of NO donors. In isolated guinea pig atria, the increase in HR in response to 1-100 micromol/l 3-morpholino-sydnonimine (SIN-1; with superoxide dismutase, n=6) or diethylamine-NO (DEA-NO, n=8) was significantly attenuated by blockers of the cGMP-inhibited phosphodiesterase (PDE3; trequinsin, milrinone or Ro-13-6438, n=22). In addition, the rate response to DEA-NO or sodium nitroprusside (SNP) was significantly reduced following inhibition of PKA (KT5720 or H-89, n=15) but not PKG (KT5728 or Rp-8-pCPT-cGMPs, n=16). Suppression of sarcoplasmic (SR) Ca(2+)release by pretreatment of isolated atria with ryanodine or cyclopiazonic acid (2 micromol/l and 60 micromol/l, n=16) significantly reduced the chronotropic response to 1-100 micromol/l SIN-1 or DEA-NO. Moreover, in isolated guinea pig sinoatrial node cells 5 micromol/l SNP significantly increased diastolic and peak Ca(2+)fluorescence (+13+/-1% and +28+/-1%, n=6, P<0.05). Our findings are consistent with a functionally significant role of cAMP/PKA signaling (via cGMP inhibition of PDE3) and SR Ca(2+)in mediating the positive chronotropic effect of NO donors.     

Inhibition of type 4 phosphodiesterase by rolipram and Ginkgo biloba extract (EGb 761) decreases agonist-induced rises in internal calcium in human endothelial cells

The effects of Gingko biloba extract EGb 761 on 5 isolated, vascular, cyclic nucleotide phosphodiesterase (PDE) isoforms were evaluated. EGb 761 preferentially inhibited PDE4 (IC(50)=25.1 mg/L), the isoform that is mainly present in endothelial cells, in a competitive manner (K:(i)=12.5 mg/L). Because changes in cyclic nucleotide levels may affect intracellular calcium ([Ca(2+)](i)) levels in endothelial cells, we examined the effects of EGb 761 on both resting [Ca(2+)](i) levels and agonist-induced rises in [Ca(2+)](i) in single human umbilical vein endothelial cells (HUVECs) in culture. The effects of EGb 761 were compared with those of rolipram, a selective PDE4 inhibitor that increases cellular cAMP levels, and the cAMP analogue dibutyryl cAMP (db-cAMP). EGb 761 (20 and 100 mg/L), rolipram (50 micromol/L), and db-cAMP (100 micromol/L) significantly inhibited histamine-, ATP-, and thrombin-induced [Ca(2+)](i) increases in HUVECs without modifying resting [Ca(2+)](i) levels. Similar results were obtained by using a Ca(2+)-free bath solution. EGb 761 (100 mg/L), but not rolipram (50 micromol/L) or db-cAMP (100 micromol/L), also inhibited Ca(2+) influx into cells having thapsigargin-depleted internal Ca(2+) stores and bathed in a Ca(2+)-free external solution. Our results are consistent with an inhibition of PDE activity that causes a reduction of agonist-induced increases in [Ca(2+)](i) in HUVECs, mainly by inhibition of Ca(2+) mobilization from internal stores. It thus may be that the cardiovascular effects of EGb 761 involve inhibition of PDE4 activity and subsequent modification of Ca(2+) signaling in endothelial cells.     

Decreased intracellular calcium stimulates renin release via calcium-inhibitable adenylyl cyclase

Intracellular calcium and cAMP are the 2 second messengers that regulate renin release; cAMP stimulates renin release from juxtaglomerular (JG) cells, whereas increased intracellular calcium inhibits it. We hypothesized that decreased intracellular calcium acts by activating calcium-inhibitable isoforms of adenylyl cyclase, increasing cAMP, and stimulating renin secretion. We used a primary culture of JG cells isolated from C-57/B6 mice. Cells were plated to a density of 70% in serum-free medium and incubated for 2 hours with or without 100 micromol/L of the cytosolic calcium chelator 5'5-dimethyl-1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid (BAPTA-AM) to decrease intracellular calcium. JG cell cAMP content and renin release were determined by radioimmunoassay. Intracellular cAMP content was 4.04+/-0.92 pM/mL per milligram of protein, and it increased by125+/-33% (P<0.01) with BAPTA-AM. Basal renin was 1.28+/-0.40 microg of angiotensin I per milliliter per hour per milligram of protein, and BAPTA-AM increased it by 182+/-62% (P<0.025). Western blots using an antibody that recognizes adenylyl cyclase types V and VI yielded a characteristic band of approximately 135 kDa. When primary cultures of isolated JG cells were tested for the calcium-inhibitable isoforms of adenylyl cyclase, they showed intense focal cytoplasmic staining. Cells stained for both renin and adenylyl cyclase V/VI showed colocalization in the cytoplasm, primarily on the granules. An adenylyl cyclase inhibitor (SQ 22,536) completely blocked BAPTA-AM-stimulated renin release and JG cell cAMP content. We conclude that calcium-inhibitable isoform(s) of adenylyl cyclase (types V and/or VI) exist within the JG cell. Thus, decreased intracellular calcium stimulates adenylyl cyclase, resulting in cAMP synthesis and, consequently, renin release.     

Direct demonstration of exocytosis and endocytosis in single mouse juxtaglomerular cells

The rate of renin secretion from renal juxtaglomerular (JG) cells is the major determinant of the activity of the renin-angiotensin system. However, the mechanisms involved in the excretion and turnover of secretory granules in the JG cells remain obscure. Therefore, in the present study, the whole-cell patch-clamp technique was applied to single JG cells from the mouse kidney to measure changes in cell membrane capacitance (Cm) as an index of secretory activity. Resting JG cell Cm was stable, on average 3. 13+/-0.13 pF (SEM, n=106). In isotonic solutions, Cm was unaffected by [Cl-]i. Cm was consistently increased (7.0+/-1.3% and 7.2+/-3.1%) by intracellular cAMP (1 to 10 micromol/L). This effect was mimicked by extracellular application of the beta-agonist isoproterenol to the JG cells (9.4+/-3.1%). At 100 micromol/L, cAMP induced a paradoxical decrease in Cm of 相似文献   

磷酸二酯酶抑制剂与呼吸道疾病

磷酸二酯酶(PDE)存在于许多炎症细胞及结构细胞中,目前已发现11种.PDE抑制剂主要抑制体内环磷酸腺苷(cAMP)及环磷酸鸟苷(cGMP)水解,使细胞内cAMP及cGMP浓度增加,引起一系列生理功能,如平滑肌舒张、减轻细胞炎症及免疫反应等.PDE4特异性水解cAMP,选择性PDE4抑制剂具有广泛抗炎作用,如抑制细胞趋化,抑制中性粒细胞、嗜酸粒细胞、巨噬细胞及T细胞细胞因子及化学趋化物质释放.第二代PDE4抑制剂Cilomilast和Roflumilast已进入临床实验阶段,并已证实对支气管哮喘(简称哮喘)及慢性阻塞性肺疾病(chronic obstructive pulmonary disease,COPD)有效.由于胃肠道副作用,这类药物临床应用受到一定限制.PDE5可特异性水解cGMP,对缺氧性肺动脉高压和血管重塑有效.PDE3和PDE7特异性水解cAMP,PDE7参与T细胞激活.目前其他PDE抑制剂与PDE4抑制剂混合制剂正在研发中.PDE4-PDE7双重抑制剂可能对哮喘及COPD更有效.PDE3-PDE4双重抑制剂具有更强的支气管舒张作用及气道保护作用.     

The calcium paradoxon of renin release: calcium suppresses renin exocytosis by inhibition of calcium-dependent adenylate cyclases AC5 and AC6

An increase in the free intracellular calcium concentration promotes exocytosis in most secretory cells. In contrast, renin release from juxtaglomerular (JG) cells is suppressed by calcium. The further downstream signaling cascades of this so called "calcium paradoxon" of renin secretion have been incompletely defined. Because cAMP is the main intracellular stimulator of renin release, we hypothesized that calcium might exert its suppressive effects on renin secretion via the inhibition of the calcium-regulated adenylate cyclases AC5 and AC6. In primary cultures of JG cells, calcium-dependent inhibitors of renin release (angiotensin II, endothelin-1, thapsigargin) suppressed renin secretion, which was paralleled by decreases in intracellular cAMP levels [cAMP]. When [cAMP] was clamped by membrane permeable cAMP derivates, renin release was not suppressed by any of the calcium liberators. Additionally, both endothelin and thapsigargin suppressed cAMP levels and renin release in isoproterenol or forskolin-pretreated As4.1 cells, a renin-producing cell line that expresses AC5 and AC6. The calcium-dependent inhibition of intracellular cAMP levels and renin release was prevented by small interfering RNA-mediated knockdown of AC5 and/or AC6 expression, underlining the functional significance of these AC isoforms in renin-producing cells. Finally, in isolated perfused mouse kidneys, angiotensin II completely inhibited the stimulation of renin secretion induced by adenylate cyclase activation (isoproterenol) but not by membrane permeable cAMP analogs, supporting the conclusion that the suppressive effect of calcium liberators on renin release is mediated by inhibition of adenylate cyclase activity.     

Regulation by phosphodiesterase isoforms of protein kinase A-mediated attenuation of myocardial protein kinase D activation

Protein kinase D (PKD) targets several proteins in the heart, including cardiac troponin I (cTnI) and class II histone deacetylases, and regulates cardiac contraction and hypertrophy. In adult rat ventricular myocytes (ARVM), PKD activation by endothelin-1 (ET1) occurs via protein kinase Cε and is attenuated by cAMP-dependent protein kinase (PKA). Intracellular compartmentalisation of cAMP, arising from localised activity of distinct cyclic nucleotide phosphodiesterase (PDE) isoforms, may result in spatially constrained regulation of the PKA activity that inhibits PKD activation. We have investigated the roles of the predominant cardiac PDE isoforms, PDE2, PDE3 and PDE4, in PKA-mediated inhibition of PKD activation. Pretreatment of ARVM with the non-selective PDE inhibitor isobutylmethylxanthine (IBMX) attenuated subsequent PKD activation by ET1. However, selective inhibition of PDE2 [by erythro-9-(2-hydroxy-3-nonyl) adenine, EHNA], PDE3 (by cilostamide) or PDE4 (by rolipram) individually had no effect on ET1-induced PKD activation. Selective inhibition of individual PDE isoforms also had no effect on the phosphorylation status of the established cardiac PKA substrates phospholamban (PLB; at Ser16) and cTnI (at Ser22/23), which increased markedly with IBMX. Combined administration of cilostamide and rolipram, like IBMX alone, attenuated ET1-induced PKD activation and increased PLB and cTnI phosphorylation, while combined administration of EHNA and cilostamide or EHNA and rolipram was ineffective. Thus, cAMP pools controlled by PDE3 and PDE4, but not PDE2, regulate the PKA activity that inhibits ET1-induced PKD activation. Furthermore, PDE3 and PDE4 play redundant roles in this process, such that inhibition of both isoforms is required to achieve PKA-mediated attenuation of PKD activation.     

A biochemical and functional assessment of monocyte phosphodiesterase activity in healthy and asthmatic subjects

The aim of this study was to investigate whether cyclic adenosine 3'5-monophosphate (cAMP) phosphodiesterase (PDE) activity is altered in monocytes from mild asthmatic subjects. Total cAMP PDE activity (pmol/min per mg protein) was significantly greater in homogenates prepared from monocytes from asthmatic subjects (68.3 +/- 7.0, n=9) compared to healthy individuals (46.3 +/- 3.3, n=14, P<0.05). The PDE inhibitors siguazodan (PDE3-selective), rolipram (PDE4-selective) and theophylline (non-selective) produced a concentration-dependent inhibition of cAMP PDE activity in homogenates from monocytes from normal and asthmatic subjects. However, siguazodan produced significantly greater (P<0.05), and rolipram significantly less (P<0.05), inhibition of total cAMP PDE activity in monocytes from asthmatics (n=4) than from healthy individuals (n=5). cAMP PDE activity was inhibited with equal potency by theophylline in monocytes from healthy and asthmatic subjects. We also investigated the functional consequences of the changes in PDE activity in mononuclear cells obtained from asthmatic subjects. There was no significant difference in the ability of PDE4 inhibitors to attenuate TNF alpha release from monocytes obtained from asthmatic compared with healthy subjects (P>0.05). Despite a significant increase in the biochemical activity of PDE3 in monocytes from asthmatic subjects, the PDE3 inhibitor siguazodan, failed to significantly reduce TNF alpha release from human monocytes. Thus, total cAMP PDE activity is increased in monocytes taken from mild asymptomatic asthmatics compared to healthy subjects and is reflected by an increase in the proportion of PDE3 and a decrease in the proportion of PDE4. This augmented enzyme activity was not associated with an alteration in the ability of PDE4 inhibitors to attenuate mononuclear cell function from asthmatics compared to healthy individuals.     

Interaction between cyclic nucleotide second messenger systems in murine Leydig cells.

Mouse Leydig cell androgen production can be acutely stimulated by atrial natriuretic factor (ANF) via cyclic guanosine 3',5'-monophosphate (cGMP). This stimulation can approach that seen with high concentrations of luteinizing hormone (LH) acting via cyclic adenosine 3',5'-monophosphate (cAMP). To assess the potential for synergistic interaction between LH/cAMP and ANF/cGMP Leydig cells were co-exposed to ANF and LH or ANF/cGMP and site/type-selective cAMP analogues. Co-exposure to 1 nM ANF and 1 ng/ml LH elicited a synergistic increase in androgen production. Both 500 microM 8-bromo-cGMP and ANF (1.0-2.5 nM) synergized with cAMP analogues selective for either of the two major isoenzymes of protein kinase A. Phosphodiesterase (PDE) inhibition was not involved as inclusion of a PDE inhibitor only augmented the response. It appears that ANF/cGMP may interact cooperatively with LH/cAMP in the stimulatory control of androgen production in the mouse Leydig cell and that the site of synergistic interaction may be the activation of the cAMP-dependent protein kinase.     

Phosphodiesterase 4 inhibition synergizes with relaxin signaling to promote decidualization of human endometrial stromal cells

The decidualization of endometrial stromal cells in the secretory phase of the menstrual cycle is an essential prerequisite for the implantation of a blastocyst. This profound differentiation process is accompanied by sustained elevated intracellular cAMP concentrations in vivo. Primary cell cultures of endometrial stromal cells decidualize by treatment with cAMP-elevating agents in vitro. Our previous findings indicated that the cAMP-degrading activities of phosphodiesterases (PDE) and signaling of the peptide hormone relaxin are coupled in human endometrial stromal cells. In the present study we have chosen a pharmacological approach to test whether relaxin binding and PDE inhibition cooperate to induce decidualization. Measurement of PDE activity and relaxin-stimulated cAMP accumulation in the presence of diverse PDE inhibitors identified PDE4 and PDE8 as the principal PDE isoforms involved in human endometrial stromal cells. The PDE4 inhibitor rolipram was most effective in elevating intracellular cAMP concentrations and synergizing with relaxin to achieve maximal in vitro decidualization, as determined by measurement of the expression of the decidual marker genes for prolactin and IGF-binding protein-1 and measurement of prolactin secretion. Gene expression for PDE4D and PDE4C was significantly up-regulated during in vitro decidualization. Treatment of cell cultures with the protein kinase A inhibitor H89 revealed a minor role for protein kinase A-mediated positive feedback control of PDE4 activity in human endometrial stromal cells, consistent with sustained elevated cAMP essential for decidualization in vitro. These findings introduce the new idea of clinically applying the combination of a specific PDE4 inhibitor with an effector such as relaxin, thereby offering an alternative nonsteroidal luteal phase support for the endometrium to encourage endometrial development and implantation in subfertile women undergoing assisted reproductive technology procedures.     

Differential regulation of endothelial cell permeability by cGMP via phosphodiesterases 2 and 3

Endothelial barrier dysfunction leading to increased permeability and vascular leakage is an underlying cause of several pathological conditions, including edema and sepsis. Whereas cAMP has been shown to decrease endothelial permeability, the role of cGMP is controversial. Endothelial cells express cGMP-inhibited phosphodiesterase (PDE)3A and cGMP-stimulated PDE2A. Thus we hypothesized that the effect of cGMP on endothelial permeability is dependent on the concentration of cGMP present and on the relative expression levels of PDE2A and PDE3A. When cAMP synthesis was slightly elevated with a submaximal concentration of 7-deacetyl-7-(O-[N-methylpiperazino]-gamma-butyryl)-dihydrochloride-forskolin (MPB-forskolin), we found that low doses of either atrial natriuretic peptide (ANP) or NO donors potentiated the inhibitory effects of MPB-forskolin on thrombin-induced permeability. However, this inhibitory effect of forskolin was reversed at higher doses of ANP or NO. These data suggest that cGMP at lower concentrations inhibits PDE3A and thereby increases a local pool of cAMP, whereas higher concentrations cGMP activates PDE2A, reversing the effect. Inhibitors of PDE3A mimicked the effect of low-dose ANP on thrombin-induced permeability, and inhibition of PDE2A reversed the stimulation of permeability seen with higher doses of ANP. Finally, increasing PDE2A expression with tumor necrosis factor-alpha reversed the inhibition of permeability caused by low doses of ANP. As predicted, the effect of tumor necrosis factor-alpha on permeability was reversed by a PDE2A inhibitor. These findings suggest that the effect of increasing concentrations of cGMP on endothelial permeability is biphasic, which, in large part, is attributable to the relative amounts of PDE2A and PDE3A in endothelial cells.     

Spatiotemporal dynamics of beta-adrenergic cAMP signals and L-type Ca2+ channel regulation in adult rat ventricular myocytes: role of phosphodiesterases

Steady-state activation of cardiac beta-adrenergic receptors leads to an intracellular compartmentation of cAMP resulting from localized cyclic nucleotide phosphodiesterase (PDE) activity. To evaluate the time course of the cAMP changes in the different compartments, brief (15 seconds) pulses of isoprenaline (100 nmol/L) were applied to adult rat ventricular myocytes (ARVMs) while monitoring cAMP changes beneath the membrane using engineered cyclic nucleotide-gated channels and within the cytosol with the fluorescence resonance energy transfer-based sensor, Epac2-camps. cAMP kinetics in the two compartments were compared to the time course of the L-type Ca(2+) channel current (I(Ca,L)) amplitude. The onset and recovery of cAMP transients were, respectively, 30% and 50% faster at the plasma membrane than in the cytosol, in agreement with a rapid production and degradation of the second messenger at the plasma membrane and a restricted diffusion of cAMP to the cytosol. I(Ca,L) amplitude increased twice slower than cAMP at the membrane, and the current remained elevated for approximately 5 minutes after cAMP had already returned to basal level, indicating that cAMP changes are not rate-limiting in channel phosphorylation/dephosphorylation. Inhibition of PDE4 (with 10 micromol/L Ro 20-1724) increased the amplitude and dramatically slowed down the onset and recovery of cAMP signals, whereas PDE3 blockade (with 1 micromol/L cilostamide) had a minor effect only on subsarcolemmal cAMP. However, when both PDE3 and PDE4 were inhibited, or when all PDEs were blocked using 3-isobutyl-l-methylxanthine (300 micromol/L), cAMP signals and I(Ca,L) declined with a time constant >10 minutes. cAMP-dependent protein kinase inhibition with protein kinase inhibitor produced a similar effect as a partial inhibition of PDE4 on the cytosolic cAMP transient. Consistently, cAMP-PDE assay on ARVMs briefly (15 seconds) exposed to isoprenaline showed a pronounced (up to approximately 50%) dose-dependent increase in total PDE activity, which was mainly attributable to activation of PDE4. These results reveal temporally distinct beta-adrenergic receptor cAMP compartments in ARVMs and shed new light on the intricate roles of PDE3 and PDE4.     

Synthetic rat atrial natriuretic factor inhibits in vitro and in vivo renin secretion in rats

We investigated the action of a synthetic rat atrial natriuretic factor (ANF) with 28 amino acids on renin secretion in rats. Renin release by kidney cortex slices was determined after 90 min of incubation at 37C. ANF inhibited basal renin release in a dose-related fashion. ANF also decreased cAMP release and increased cGMP release in a dose-dependent manner. Renin release stimulated by 10(-7) M isoproterenol was inhibited by ANF with an ID50 of 5.8 x 10(-8) M. The renin-inhibitory effect was not calcium-dependent. In anesthetized rats, a bolus IV dose of ANF decreased plasma renin activity and cAMP concentration, but increased cGMP concentration. These data suggest that ANF inhibits renin secretion via the direct action on juxtaglomerular cells and that this effect may be partly mediated by the changes in cyclic nucleotide production.     

Mechanism of SNAP potentiating antiproliferative effect of calcitonin gene-related peptide in cultured vascular smooth muscle cells.

We previously showed that CGRP inhibits cell proliferation which correlates with an elevation of cAMP levels in rabbit aortic vascular smooth muscle cells (VSMCs). The present study determined the effects of S-nitroso-N-acetylpenicillamine (SNAP, a nitric oxide donor) on CGRP-induced antiproliferative effects and cellular mechanism in cultured rabbit aortic VSMCs. The cells (in fifth-sixth passage) were exposed to 2.5% fetal bovine serum for 24 h in the presence or absence of SNAP, hCGRP or both.(3)H-thymidine incorporation was used to measure DNA synthesis. The results showed that SNAP (60-100 microm) significantly inhibited the proliferation and elevated cGMP levels in cultured rabbit aortic VSMCs. In combination, however, SNAP (30 microm) potentiated hCGRP (10-100 n m)-induced antiproliferation. SNAP (30 microm) and hCGRP (10-100 n m) or forskolin (10 microm), an activator of adenylate cyclase, caused more than additive cAMP elevations, but not cGMP elevations, in these cells. Quazinone, an inhibitor of cGMP-inhibited-phosphodiesterase (cGI-PDE, PDE3), or SNAP plus quazinone caused a similar potentiation as SNAP of the hCGRP-induced elevations of cAMP levels. The data indicate that SNAP-induced potentiation of CGRP's effects likely involves inhibition of cGI-PDE, thus allowing enhanced accumulation of cAMP that mediates the antiproliferative effects of hCGRP in cultured rabbit aortic VSMCs.     

Platelet cyclic adenosine monophosphate phosphodiesterases: targets for regulating platelet-related thrombosis

Platelets contain two cyclic adenosine monophosphate (cAMP) phosphodiesterases (PDEs) that regulate the level of cAMP, the major inhibitor of platelet activation pathways. PDE3A hydrolyzes cAMP to 5' AMP with a low K (m). PDE3A is inhibited by cyclic guanosine monophosphate (cGMP), which provides a feedback control and controls basal levels of cAMP. In contrast, PDE2A hydrolyzes both cAMP and cGMP with a high K (m), is allosterically stimulated by cGMP at moderate levels, and may control the stimulated levels of cAMP. Using affinity labeling, chemical modification, and site-directed mutagenesis of highly conserved amino acids, the amino acids required for catalytic activity and/or metal binding are H752 and H756. The singular binding sites for cAMP include N845, E971, and F972, whereas the unique amino acids interacting with cGMP are Y751, H836, H849, and D950. Residues E866 and F1004 are present in both the overlapping cGMP and cAMP sites. Two inhibitors of PDE3A are used in clinical medicine: milrinone and cilostazol. Three amino acids, Y751, D950, and F1004, show decreased sensitivity to both inhibitors (increased K (i)). These inhibitors mimic cGMP as an inhibitor of PDE3A rather than compete for cAMP binding. New nonhydrolyzable affinity labels inactivate PDE3A and are protected by Sp-cAMPS, a nonhydrolyzable substrate of the enzyme. These compounds have the potential to identify amino acids that are unique for PDE3A. An inhibitor of platelet PDE2A increases cAMP more than inhibitors of PDE3A but has much less effect on platelet activation, suggesting that these enzymes are present in different compartments of the cell.     

Responses of juxtaglomerular cell suspensions to various stimuli

Cell suspensions were prepared from rat renal cortical tissue by dispersion with 0.1% collagenase. Unit gravity sedimentation in a 1%-4% Ficoll gradient resulted in a single-cell suspension enriched in juxtaglomerular (JG) cells. Both the cellular renin activity and the amount of renin released into the supernatant increased with time when the suspensions were incubated for 1 hour at 37 degrees C in tissue culture medium. These cells responded to epinephrine and norepinephrine by increasing both synthesis and release of renin. The response was blocked by timolol but not by phenoxybenzamine. Cell suspensions prepared in the same manner but using 0.25% trypsin as the dispersing enzyme neither synthesized nor released renin into the tissue culture medium when similarly incubated. Trypsin-dispersed cells did not respond to catecholamine stimulation. Renin synthesis and release in collagenase-dispersed JG cells were unaltered by changes in Na, K, or Ca ion concentrations. Angiotensin II inhibited release, while saline extracts of clipped kidney from renal hypertensive rats stimulated renin release by these cells.