cGMP Phosphodiesterase Inhibitor Research Articles

Vasodilator responses to ATP and UTP are cAMP dependent in the mesenteric vascular bed of the cat.

This study was designed to examine the responses to and the mechanism by which purinergic agonists decrease vascular resistance in the mesenteric vascular bed of the cat. Injections of ATP, UTP, and 2-MethylThioATP (2-MetSATP) into the mesenteric perfusion circuit elicited dose-dependent decreases in perfusion pressure while injections of beta,gamma-MethylATP (beta,gamma-MetATP) produced a biphasic response with an initial vasopressor response followed by a vasodilator response. The order of potency of the vasodilator response was 2-MetSATP > ATP > UTP > beta,gamma-MetATP. The vasodilator responses to ATP, UTP, 2-MetSATP, and beta,gamma-MetATP were increased in duration by the cAMP phosphodiesterase inhibitor, rolipram. However, vasodilator responses were not altered by the adminstration of a nitric oxide synthase inhibitor, a cGMP phosphodiesterase inhibitor, or a cyclooxygenase inhibitor. Treatment with PPADS, a P2X(1), P2Y(1), and P2Y(4) receptor antagonist, did not alter vasodilator responses to the purinergic agonists; however, the vasopressor component of the response to beta,gamma-MetATP was decreased. These data suggest that ATP, UTP, 2-MetSATP, and beta,gamma-MetATP dilate the mesentary vascular bed in the cat by a cAMP dependent mechanism, and that nitric oxide or prostaglandin release, cGMP accumulation, or activation of P2X(1), P2Y(1), or P2Y(4) receptors play little or no role in mediating vasodilator responses to the purinergic agonists in this regional vascular bed. In addition, these results suggest that the pressor component of the response to beta,gamma-MetATP is mediated by the activation of P2X(1) receptors.

Novel selective PDE4 inhibitors. 1. Synthesis, structure-activity relationships, and molecular modeling of 4-(3,4-dimethoxyphenyl)-2H-phthalazin-1-ones and analogues.

A number of 6-(3,4-dimethoxyphenyl)-4,5-dihydro-2H-pyridazin-3-ones and a novel series of 4-(3,4-dimethoxyphenyl)-2H-phthalazin-1-ones were prepared and tested on the cGMP-inhibited phosphodiesterase (PDE3) and cAMP-specific phosphodiesterase (PDE4) enzymes. All tested compounds were found to specifically inhibit PDE4 except for pyridazinone 3b, which showed moderate PDE4 (pIC(50) = 6.5) as well as PDE3 (pIC(50) = 6.6) inhibitory activity. In both the pyridazinone and phthlazinone series it was found that N-substitution is beneficial for PDE4 inhibition, whereas in the pyridazinone series it also accounts for PDE4 selectivity. In the phthalazinone series, the cis-4a,5,6,7,8,8a-hexahydrophthalazinones and their corresponding 4a,5,8,8a-tetrahydro analogues showed potent PDE4 inhibitory potency (10/11c,d: pIC(50) = 7.6-8.4). A molecular modeling study revealed that the cis-fused cyclohexa(e)ne rings occupy a region in space different from that occupied by the other fused (un)saturated hydrocarbon rings applied; we therefore assume that the steric interactions of these rings with the binding site play an important role in enzyme inhibition.

Cyclic GMP regulation of the L-type Ca(2+) channel current in human atrial myocytes.

1. The regulation of the L-type Ca(2+) current (I(Ca)) by intracellular cGMP was investigated in human atrial myocytes using the whole-cell patch-clamp technique. 2. Intracellular application of 0.5 microM cGMP produced a strong stimulation of basal I(Ca) (+64 +/- 5 %, n = 60), whereas a 10-fold higher cGMP concentration induced a 2-fold smaller increase (+36 +/- 8 %, n = 35). 3. The biphasic response of I(Ca) to cGMP was not mimicked by the cGMP-dependent protein kinase (PKG) activator 8-bromoguanosine 3',5' cyclic monophosphate (8-bromo-cGMP, 0.5 or 5 microM), and was not affected by the PKG inhibitor KT 5823 (100 nM). 4. In contrast, cGMP stimulation of I(Ca) was abolished by intracellular perfusion with PKI (10 microM), a selective inhibitor of the cAMP-dependent protein kinase (PKA). 5. Selective inhibition of the cGMP-inhibited phosphodiesterase (PDE3) by extracellular cilostamide (100 nM) strongly enhanced basal I(Ca) in control conditions (+78 +/- 13 %, n = 7) but had only a marginal effect in the presence of intracellular cGMP (+22 +/- 7 % in addition to 0.5 microM cGMP, n = 11; +20 +/- 22 % in addition to 5 microM cGMP, n = 7). 6. Application of erythro-9-[2-hydroxy-3-nonyl]adenine (EHNA, 30 microM), a selective inhibitor of the cGMP-stimulated phosphodiesterase (PDE2), fully reversed the secondary inhibitory effect of 5 microM cGMP on I(Ca) (+99 +/- 16 % stimulation, n = 7). 7. Altogether, these data indicate that intracellular cGMP regulates basal I(Ca) in human atrial myocytes in a similar manner to NO donors. The effect of cGMP involves modulation of the cAMP level and PKA activity via opposite actions of the nucleotide on PDE2 and PDE3.

N-acetyl-L-cysteine exerts direct anti-aggregating effect on human platelets.

N-acetyl-L-cysteine, a thiol compound, has been shown to potentiate the inhibition of platelet aggregation exerted by organic nitrates and to increase the anti-aggregating effect of L-arginine, which promotes endogenous synthesis of nitric oxide (NO) acting as substrate of platelet constitutive nitric oxide synthase (NOS). It is not known whether this thiol can exert direct effects on platelet aggregability. 14 healthy male volunteers provided platelet samples to investigate whether N-acetyl-L-cysteine directly influences platelet function and intraplatelet levels of 3',5' cyclic guanosine monophosphate (cGMP), which represents the second messenger involved in NO-induced antiaggregation. Some experiments were repeated in the presence of NOS inhibitor NG-monomethyl-L-arginine (L-NMMA), of nitric oxide-sensitive guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), of the selective cGMP phosphodiesterase inhibitor zaprinast and of calcium ionophores (A23187, ionomycin). N-acetyl-L-cysteine at 3000-6000 micromol L-1 decreases the responses of human platelets both in platelet-rich plasma (aggregation induced by adenosine 5-diphosphate) and in whole blood (aggregation induced by collagen). The anti-aggregating effect was prevented by preincubation with L-NMMA and guanylyl cyclase inhibitor ODQ. In resting platelets, N-acetyl-L-cysteine increased the levels of cGMP starting from a concentration of 3000 micromol L-1. Permeabilized platelets exhibited an increased sensitivity to the anti-aggregating effect of N-acetyl-L-cysteine. Also, cGMP phosphodiesterase inhibition or the increase in calcium availability, enhanced N-acetyl-L-cysteine effects on platelets. N-acetyl-L-cysteine exerts direct anti-aggregating effects through an increased bioavailability of platelet nitric oxide.

Stimulation of Nitric Oxide–cGMP Pathway Excites Striatal Cholinergic Interneurons via Protein Kinase G Activation

Conflicting data have been collected so far on the action of nitric oxide (NO) on cholinergic interneurons of the striatum. In the present in vitro electrophysiological study, we reported that intracellularly recorded striatal cholinergic interneurons are excited by both hydroxylamine and S-nitroso-N-acetylpenicillamine, two NO donors. This excitation persisted unchanged in the presence of glutamate, dopamine, and substance P receptor antagonists as well as after blockade of tetrodotoxin (TTX)- and calcium channel-sensitive transmitter release, suggesting that NO produces its effects by modulating directly resting ion conductances in the somatodendritic region of striatal cholinergic cells. The depolarizing effect of hydroxylamine was greatly reduced by lowering external concentrations of sodium ions (from 126 to 38 mm) and did not reverse polarity in the voltage range from -120 to -40 mV. The sodium transporter blockers bepridil and 3',4'-dichlorobenzamil were conversely ineffective in preventing NO-induced membrane depolarization. Intracellular cGMP elevation is required for the action of hydroxylamine on striatal cholinergic cells, as demonstrated by the findings that the membrane depolarization produced by this pharmacological agent was prevented by bath and intracellular application of two inhibitors of soluble guanylyl cyclase and was mimicked and occluded by zaprinast, a cGMP phosphodiesterase inhibitor. Finally, intracellular Rp-8-Br-cGMPS, a protein kinase G (PKG) inhibitor, blocked the hydroxylamine-induced membrane depolarization of cholinergic interneurons, whereas both okadaic acid and calyculin A, two protein phosphatase inhibitors, enhanced it, indicating that intracellular PKG and phosphatases oppositely regulate the sensitivity of striatal cholinergic interneurons to NO. The characterization of the cellular mechanisms involved in the regulation of striatal interneuron activity is a key step for the understanding of the role of these cells in striatal microcircuitry.

Upregulation of angiotensin-converting enzyme by vascular endothelial growth factor.

The role of vascular endothelial growth factor (VEGF), a potent endothelium-specific angiogenic factor, in the regulation of angiotensin-converting enzyme (ACE) in cultured human umbilical vein endothelial cells (HUVECs) was studied. VEGF (0.07-1.2 x 10(-6) mmol/l) caused a dose-dependent increase in ACE measured in intact endothelial cells and increased the expression of ACE mRNA. The stimulatory effect of VEGF was inhibited by pretreatment of endothelial cells with the tyrosine kinase inhibitor herbimycin (4.35 x 10(-5) mmol/l). The stimulatory effect of VEGF was potentiated by the selective cGMP phosphodiesterase inhibitor zaprinast (0.1 mmol/l). The nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME; 5.4 mmol/l) suppressed the stimulatory effect of VEGF. The nonselective cyclooxygenase (COX) inhibitor indomethacin (5 microM) and the selective COX-2 inhibitor NS-398 (5 microM) potentiated the stimulatory effect of VEGF, whereas the selective COX-1 inhibitor resveratrol (5 microM) was without effect. ACE induction by VEGF was inhibited by the selective protein kinase C (PKC) inhibitor GF109203X (2.5 x 10(-3) mmol/l) and by downregulating PKC with phorbol 12-myristate 13-acetate. In summary, VEGF induced ACE in cultured HUVECs. Intracellular events such as tyrosine kinase activation, PKC activation, and increase of cGMP were probably involved in ACE induction by VEGF. Nitric oxide may partially contribute to ACE induction by VEGF. The powerful capacity of VEGF to increase ACE in endothelial cells shown here suggests a synergistic relation between VEGF and the renin-angiotensin system in vascular biology and pathophysiology.

Relaxation induced by cGMP phosphodiesterase inhibitors sildenafil and zaprinast in human vessels

Background. Sildenafil is currently used in the treatment of erectile dysfunction. However, assessment of direct effects of sildenafil on coronary arteries and on arteries used as coronary grafts is unknown. This study was designed to investigate the effects of sildenafil on contracted human coronary, internal mammary, and radial arteries obtained from multiorgan donors. The observations were extended to forearm veins. Zaprinast was included in this study for comparison. Methods. Segments of left coronary, internal mammary, and radial arteries, and forearm veins were obtained from 16 multiorgan donors. Vascular rings were suspended in organ bath chambers and isometric tension was recorded. Then the effects of sildenafil, zaprinast, and sodium nitroprusside on precontracted vessels were studied. Results. Sildenafil (10 −8 − 3 × 10 −5 mol/L) caused concentration-dependent relaxation in the internal mammary arteries, radial arteries, and forearm veins. In the coronary arteries, sildenafil had a modest relaxant effect. In addition, sildenafil amplified the relaxation induced by sodium nitroprusside in all four vessels. Relaxation was unaffected by the inhibitor of nitric oxide synthase N G-monomethyl-L-arginine (10 −4 mol/L). Compared with zaprinast, sildenafil was eight to ten times more potent in terms of EC 50 values. Conclusions. The direct relaxant effects of sildenafil together with its synergistic interaction with nitric oxide donors should be considered in patients undergoing coronary bypass surgery, patients with low blood pressure, and patients receiving antihypertensive regimes.

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 If, without affecting basal ICa-L. The activity of Ifis known to be enhanced by cyclic nucleotides and by an increase in cytosolic Ca2+. We examined the role of cGMP-dependent signaling pathways and intracelluar Ca2+stores in mediating the positive chronotropic effect of NO donors. In isolated guinea pig atria, the increase in HR in response to 1–100 μ mol/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) Ca2+release by pretreatment of isolated atria with ryanodine or cyclopiazonic acid (2 μ mol/l and 60 μ mol/l, n=16) significantly reduced the chronotropic response to 1–100 μ mol/l SIN-1 or DEA-NO. Moreover, in isolated guinea pig sinoatrial node cells 5 μ mol/l SNP significantly increased diastolic and peak Ca2+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 Ca2+in mediating the positive chronotropic effect of NO donors.

Phosphodiesterase type 5 inhibition enhances vasorelaxation caused by nitroprusside in guinea pig intact heart and isolated aorta.

Increased vascular smooth muscle cyclic guanine monophosphate (cGMP) results in vascular relaxation. The vascular effects of stimulating cGMP production with 10(-8)-10(-4) M nitroprusside (NP) and inhibiting cGMP hydrolysis with 10(-8)-10(-4) M zaprinast (ZAP), a selective type V inhibitor of cGMP phosphodiesterase (PDE), were assessed in isolated guinea pig hearts and aortic rings. Coronary flow (CF) IC50 values for NP and ZAP, respectively, were 0.8+/-0.1 x 10(-6) M and 3.6+/-0.1 x 10(-6) M; for coronary sinus pO2 IC50 values were 0.7+/-0.1 x 10(-6) M and 3.7+/-0.1 x 10(-6) M. CF increased by 13+/-2% with 10(-6) NP, and by 12+/-2% with 10(-5) M ZAP; percentage O2 extraction (%O2E) decreased by 17+/-3% with NP and 28+/-4% with ZAP. Together, 10(-6) M NP + 10(-5) M ZAP augmented the increased in CF to 23+/-3% of control, and the decrease in percentage O2 extraction (%O2E) to 40+/-4% of control. Other cardiac effects of NP and ZAP were minimal. In norepinephrine preconstricted aortic rings, the IC50 for relaxation was elicited at 0.4+/-0.1 x 10(-6) M NP and 6.1+/-0.1 x 10(-6) M ZAP. NP given with ZAP gave a logarithmic relation so that IC50 [NP] = -(57 log10 [ZAP]) + 416; R2 = 0.95. NP, 3 x 10(-7) M; ZAP, 3 x 10(-6) M; and NP + ZAP combined increased aortic tissue cGMP by eight-, nine-, and 15-fold, respectively. Inhibiting cGMP hydrolysis may be an effective approach to augment vasorelaxation elicited by cGMP synthesis in the heart.

Regulation of pacemaker currents in interstitial cells of Cajal from murine small intestine by cyclic nucleotides.

1. Electrical rhythmicity (slow waves) in gastrointestinal muscles (GI) is generated by interstitial cells of Cajal (ICC). Cultured ICC from the murine small intestine were studied with the patch-clamp technique to characterize regulation of pacemaker currents by cyclic nucleotides. Cyclic nucleotide agonists were also tested on intact strips of murine small intestine. 2. Nitric oxide donors slowed the frequency of pacemaker currents in a concentration-dependent manner. These effects depended on cGMP formation and were reduced by 1H-[1,2, 4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). The effects of nitric oxide donors were mimicked by membrane-permeable analogues of cGMP. The specific cGMP phosphodiesterase inhibitor zaprinast reduced the frequency of spontaneous pacemaker currents. 3. The cGMP-dependent effects on pacemaker currents were not affected by okadaic acid or KT-5823, an inhibitor of protein kinase G. 4. Forskolin, but not dideoxy forskolin, reduced the frequency of spontaneous pacemaker activity and activated a sustained outward current. The latter was likely to be due to ATP-dependent K+ channels because it was blocked by glibenclamide. 5. The effects of forskolin were not mimicked by membrane-permeable cAMP analogues. A membrane-permeable inhibitor of protein kinase A, myristoylated PKA inhibitor, and the adenylyl cyclase inhibitor SQ-22536, had no effect on responses to forskolin. 6. Responses of intact muscles to cGMP and cAMP agonists were similar to the responses of pacemaker cells. Changes in resting membrane potential and slow wave amplitude, however, were noted in intact jejunal muscles that were not observed in ICC. Differences in responses may have been due to the effects of cyclic nucleotide agonists on smooth muscle cells that would sum with responses of ICC in intact jejunal muscle strips. 7. A cGMP-dependent mechanism regulates slow wave frequency, but this occurs through direct action of cGMP not via protein phosphorylation. Regulation of pacemaker currents by cAMP-dependent mechanisms was not observed.

Vasodilator responses to ATP and UTP are not dependent on nitric oxide release, K+ATP channel activation, or the release of vasodilator prostaglandins in the hindlimb vascular bed of the cat

The effects of the purinergic agonists, ATP, ATPgammaS, UTP, and 2-Met-Thio AP, were investigated in the hindlimb vascular bed of the cat. Under constant-flow conditions, injections of the purinergic agonists into the perfusion circuit elicited dose-related decreases in perfusion pressure. The order of potency was 2-Met-Thio ATP > ATPgammaS > ATP > UTP. In contrast, injections of GTPgammaS, cAMP, UDP, and UMP had no effect. Vasodilator responses to ATP, ATPgammaS, UTP, and 2-Met-Thio ATP were increased in duration by the cAMP phosphodiesterase inhibitor rolipram, whereas the cGMP phosphodiesterase inhibitor zaprinast had no effect. Responses to the purinergic agonists were not altered by nitric oxide synthase inhibitors, K+(ATP) channel antagonists, cyclooxygenase inhibitors, or agents that interfere with the actions of the adrenergic nervous system. These data suggest that ATP, ATPgammaS, UTP, and 2-Met-Thio ATP dilate the hindlimb vascular bed by a direct cAMP-dependent mechanism, and that the release of nitric oxide, vasodilator prostaglandins, K+(ATP) channel opening, or an inhibitory effect on the adrenergic nervous system play little, if any, role in mediating or modulating responses to the purinergic agonists in the hindlimb circulation of the cat.

Natriuretic peptides: a new lipolytic pathway in human adipocytes

Atrial natriuretic peptide (ANP) receptors have been described on rodent adipocytes and expression of their mRNA is found in human adipose tissue. However, no biological effects associated with the stimulation of these receptors have been reported in this tissue. A putative lipolytic effect of natriuretic peptides was investigated in human adipose tissue. On isolated fat cells, ANP and brain natriuretic peptide (BNP) stimulated lipolysis as much as isoproterenol, a nonselective beta-adrenergic receptor agonist, whereas C-type natriuretic peptide (CNP) had the lowest lipolytic effect. In situ microdialysis experiments confirmed the potent lipolytic effect of ANP in abdominal s.c. adipose tissue of healthy subjects. A high level of ANP binding sites was identified in human adipocytes. The potency order defined in lipolysis (ANP > BNP > CNP) and the ANP-induced cGMP production sustained the presence of type A natriuretic peptide receptor in human fat cells. Activation or inhibition of cGMP-inhibited phosphodiesterase (PDE-3B) (using insulin and OPC 3911, respectively) did not modify ANP-induced lipolysis whereas the isoproterenol effect was decreased or increased. Moreover, inhibition of adenylyl cyclase activity (using a mixture of alpha(2)-adrenergic and adenosine A1 agonists receptors) did not change ANP- but suppressed isoproterenol-induced lipolysis. The noninvolvement of the PDE-3B was finally confirmed by measuring its activity under ANP stimulation. Thus, we demonstrate that natriuretic peptides are a new pathway controlling human adipose tissue lipolysis operating via a cGMP-dependent pathway that does not involve PDE-3B inhibition and cAMP production.

Phosphorylation of PDE3B by Phosphatidylinositol 3-Kinase Associated with the Insulin Receptor

Phosphatidylinositol 3-kinase mediates several actions of insulin including its antilipolytic effect. This effect is elicited by the insulin-stimulated serine phosphorylation and activation of cGMP-inhibited phosphodiesterase (PDE3B). In human adipocytes, we found that insulin differentially stimulated phosphatidylinositol 3-kinase activity; the lipid kinase activity was associated with IRS-1, whereas the serine kinase activity was associated with the insulin receptor and phosphorylated a number of proteins including p85, p110, and a 135-kDa protein identified as PDE3B. PDE3B phosphorylation was associated with enzyme activation, thus initiating the antilipolytic effect of insulin. These results show a novel pathway for intracellular signaling through the insulin receptor leading to the serine phosphorylation of key proteins involved in insulin action.

Store-operated Calcium Entry in Vascular Endothelial Cells Is Inhibited by cGMP via a Protein Kinase G-dependent Mechanism

Store-operated Ca(2+) entry in vascular endothelial cells not only serves to refill the intracellular Ca(2+) stores, but also acts to stimulate the synthesis of nitric oxide, a key vasodilatory factor. In this study, we examined the role of cGMP in regulating the store-operated Ca(2+) entry in aortic endothelial cells. Cyclopiazonic acid (CPA) and thapsigargin, two selective inhibitors of endoplasmic reticulum Ca(2+)-ATPase, were used to induce store-operated Ca(2+) entry. 8-Bromo-cGMP, an activator of protein kinase G, inhibited the CPA- or thapsigargin-induced Ca(2+) entry in a concentration-dependent manner. An inhibitor of protein kinase G, KT5823 (1 microM) or H-8 (10 microM), abolished the inhibitory action of 8-bromo-cGMP and resumed Ca(2+) entry. Addition of S-nitroso-N-acetylpenicillamine (a nitric oxide donor) or dipyridamole (a cGMP phosphodiesterase inhibitor) during CPA treatment elevated cellular cGMP levels, stimulated protein kinase G activity, and at the same time reduced Ca(2+) influx due to CPA. Patch clamp study confirmed the existence of a CPA-activated Ca(2+)-permeable channel sensitive to cGMP inhibition. These results suggest that cGMP via a protein kinase G-dependent mechanism may play a key role in the regulation of the store-operated Ca(2+) entry in vascular endothelial cells.

Cardiac Type cGMP-Inhibited Phosphodiesterase (PDE3A) Gene Structure: Similarity and Difference to Adipocyte Type PDE3B Gene

Phosphodiesterase type 3 isoforms, PDE3A and 3B, are expressed primarily in cardiovascular and adipose tissues, respectively. We previously reported a shorter transcript of 4.4-kb PDE3A which is predominantly transcribed in human placenta, whereas a full-length 7.6-kb transcript corresponding to the cardiac PDE3A cDNA has not been characterized. Due to unfortunate circumstances created by changes in PDE3 nomenclature, PDE3B gene structure previously reported used PDE3A in its title. Here, we describe PDE3A gene structure, which comprises 16 exons spanning over 130 kb on chromosome 12p12. Two PDE3 isoforms share similar gene organization, but localize to different chromosomes. The most distal transcription initiation site of the PDE3A gene is ∼1071 bases upstream of the ATG site, suggesting that exon 1 consists of 1071 and 960 bp of untranslated and translated sequences, respectively. The proximal 5′-flanking region, which does not contain TATA-like sequences, exhibited weak but significant promoter activity. Results suggest potential involvement of distal promoter/enhancer and translational regulation for expression of the 7.6-kb transcript.

L-NAME causes antinociception by stimulation of the arginine-NO-cGMP pathway.

NG-nitro-L-arginine methyl ester (L-NAME) has been used extensively as a paradigmatic inhibitor of NO synthase and has been shown to cause antinociception in several experimental models. We describe here how L-NAME produced a dose-dependent antinociceptive effect when injected intraperitoneally in the mouse after acetic acid induced writhings, or intraplantarly in the rat paw pressure hyperalgesia induced by carrageenin or prostaglandin E2. In contrast another NO synthase inhibitor, NG-monomethyl-L-arginine (L-NMMA), had no significant effect per se but inhibited L-NAME systemic induced antinociception in mice and local induced antinociception in the rat paw hyperalgesia test. D-NAME had no antinociceptive effect upon carrageenin-induced hyperalgesia. Pretreatment of the paws with two inhibitors of guanylate cyclase, methylene blue (MB) and 1H-:[1,2,4]-oxadiazolo-:[4,3-a] quinoxalin-1-one (ODQ) abolished the antinociceptive effect of L-NAME. L-Arginine and the cGMP phosphodiesterase inhibitor, MY 5445 significantly enhanced the L-NAME antinociceptive effect. The central antinociceptive effect of L-NAME was blocked by co-administration of L-NMMA, ODQ and MB. The present series of experiments shows that L-NAME, but not L-NMMA, has an antinociceptive effect. It can be suggested that L-NAME causes the antinociceptive effect by stimulation of the arginine/ NO/ cGMP pathway, since the antinociceptive effect of L-NAME can be antagonized by L-NMMA and abolished by the guanylate cyclase inhibitors (MB and ODQ). In addition, the NO synthase substrate, L-arginine and the cGMP phosphodiesterase inhibitor, MY5445 were seen to potentiate the effects of L-NAME. Thus, L-NAME used alone, has limitations as a specific inhibitor of the arginine-NO-cGMP pathway and may therefore be a poor pharmacological tool for use in characterising participation in pathophysiological processes.

C-type natriuretic peptide inhibits ANP secretion and atrial dynamics in perfused atria: NPR-B-cGMP signaling.

The purpose of the present experiments was to define the role of C-type natriuretic peptide (CNP) in the regulation of atrial secretion of atrial natriuretic peptide (ANP) and atrial stroke volume. Experiments were performed in perfused beating and nonbeating quiescent atria, single atrial myocytes, and atrial membranes. CNP suppressed in a dose-related fashion the increase in atrial stroke volume and ANP secretion induced by atrial pacing. CNP caused a right shift in the positive relationships between changes in the secretion of ANP and atrial stroke volume or translocation of the extracellular fluid (ECF), which indicates the suppression of atrial myocytic release of ANP into the paracellular space. The effects of CNP on the secretion and contraction were mimicked by 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP). CNP increased cGMP production in the perfused atria, and the effects of CNP on the secretion of ANP and atrial dynamics were accentuated by pretreatment with an inhibitor of cGMP phosphodiesterase, zaprinast. An inhibitor of the biological natriuretic peptide receptor (NPR), HS-142-1, attenuated the effects of CNP. The suppression of ANP secretion by CNP and 8-BrcGMP was abolished by a depletion of extracellular Ca(2+) in nonbeating atria. Natriuretic peptides increased cGMP production in atrial membranes with a rank order of potency of CNP > BNP > ANP, and the effect was inhibited by HS-142-1. CNP and 8-BrcGMP increased intracellular Ca(2+) concentration transients in single atrial myocytes, and mRNAs for CNP and NPR-B were expressed in the rabbit atrium. From these results we conclude that atrial ANP release and stroke volume are controlled by CNP via NPR-B-cGMP mediated signaling, which may in turn act via regulation of intracellular Ca(2+).

Differential role of cyclic GMP–dependent protein kinase II in ion transport in murine small intestine and colon

Background & Aims: The aim of this study was to determine the role of guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase (cGK) type II in intestinal fluid homeostasis under basal conditions and following exposure to cGMP-linked secretagogues, e.g., Escherichia coli heat-stable enterotoxin (STa) and guanylin. Methods: Fluid and ion transport was determined in different segments of the intestine of wild-type and cGK II–deficient mice by ligated loop assays in vivo, and by short-circuit current and isotope flux measurements in vitro. Results: Small intestinal fluid absorption in vivo was enhanced in cGK II–deficient mice under basal conditions and in the presence of STa. Furthermore, STa, guanylin, and 8-pCPT-cGMP stimulation of electrogenic anion secretion and inhibition of Na + absorption in vitro were markedly reduced in the small intestine from cGK II −/− mice but not in proximal colon. The type III phosphodiesterase inhibitor amrinone mimicked STa action in cGK II −/− mice, and also stimulated ion secretion in humans. Conclusions: This study shows that the cGMP/cGK II pathway regulates fluid homeostasis in the small intestine under basal conditions and mediates STa effects by both increasing anion secretion and inhibiting Na + absorption. It also demonstrates the presence of a cGK II–independent pathway for STa/cGMP-provoked secretion predominantly in the colon, which possibly involves a cGMP-inhibitable phosphodiesterase and/or activation of the cAMP–dependent protein kinase pathway. GASTROENTEROLOGY 2000;118:108-114

Ontogeny of Catecholamine and Adenosine Receptor-Mediated cAMP Signaling of Embryonic Red Blood Cells: Role of cGMP-Inhibited Phosphodiesterase 3 and Hemoglobin

We have previously shown that the cAMP signaling pathway controls major aspects of embryonic red blood cell (RBC) function in avian embryos (Glombitza et al, Am J Physiol 271:R973, 1996; and Dragon et al, Am J Physiol 271:R982, 1996) that are important for adaptation of the RBC gas transport properties to the progressive hypercapnia and hypoxia of later stages of avian embryonic development. Data about the ontogeny of receptor-mediated cAMP signaling are lacking. We have analyzed the response of primitive and definitive chick embryo RBC harvested from day 3 to 18 of development towards forskolin, β-adrenergic, and A2 receptor agonists. The results show a strong response of immature definitive and primitive RBC to adenosine A2 and β-adrenergic receptor agonists, which is drastically reduced in the last stage of development, coincident with the appearance of mature, transcriptionally inactive RBC. Modulation of cGMP-inhibited phosphodiesterase 3 (PDE3) has a controlling influence on cAMP accumulation in definitive RBC. Under physiological conditions, PDE3 is inhibited due to activation of soluble guanylyl cyclase (sGC). Inhibition of sGC with the specific inhibitor ODQ decreases receptor-mediated stimulation of cAMP production; this effect is reversed by the PDE3 inhibitor milrinone. sGC is acitivated by nitric oxide (NO), but we found no evidence for production of NO by erythrocyte NO-synthase. However, embryonic hemoglobin releases NO in an oxygen-linked manner that may activate guanylyl cyclase.

Ontogeny of Catecholamine and Adenosine Receptor-Mediated cAMP Signaling of Embryonic Red Blood Cells: Role of cGMP-Inhibited Phosphodiesterase 3 and Hemoglobin

AbstractWe have previously shown that the cAMP signaling pathway controls major aspects of embryonic red blood cell (RBC) function in avian embryos (Glombitza et al, Am J Physiol 271:R973, 1996; and Dragon et al, Am J Physiol 271:R982, 1996) that are important for adaptation of the RBC gas transport properties to the progressive hypercapnia and hypoxia of later stages of avian embryonic development. Data about the ontogeny of receptor-mediated cAMP signaling are lacking. We have analyzed the response of primitive and definitive chick embryo RBC harvested from day 3 to 18 of development towards forskolin, β-adrenergic, and A2 receptor agonists. The results show a strong response of immature definitive and primitive RBC to adenosine A2 and β-adrenergic receptor agonists, which is drastically reduced in the last stage of development, coincident with the appearance of mature, transcriptionally inactive RBC. Modulation of cGMP-inhibited phosphodiesterase 3 (PDE3) has a controlling influence on cAMP accumulation in definitive RBC. Under physiological conditions, PDE3 is inhibited due to activation of soluble guanylyl cyclase (sGC). Inhibition of sGC with the specific inhibitor ODQ decreases receptor-mediated stimulation of cAMP production; this effect is reversed by the PDE3 inhibitor milrinone. sGC is acitivated by nitric oxide (NO), but we found no evidence for production of NO by erythrocyte NO-synthase. However, embryonic hemoglobin releases NO in an oxygen-linked manner that may activate guanylyl cyclase.