Cyclase Activity Research Articles

Regulation of adenylyl cyclase activity in rat testes by acylated derivatives of peptide 562–572 of a luteinizing hormone receptor

One of directions of the search of hormonal signaling systems regulators is the development of peptides that correspond to the cytoplasmic regions of G-protein-coupled receptors (GPCR). Modification of the pepti- des with hydrophobic radicals increases their efficiency and selectivity. But currently is not studied as the acti- vity of the peptide depends on the localization of the hydrophobic radicals, their number and the chemical natu- re. The aim of this work was the synthesis of modified by fatty acid radicals derivatives of peptide 562-572 corresponding to the C-terminal region of luteinizing hormone receptor (LHR), and the study of regulatory ef- fects of the acylated LHR-peptides on the basal and hormone-stimulated activity of adenylyl cyclase (AC) in the rat tissues. To elucidate the effect of localization of hydrophobic radicals and their number the modification of peptide 562-572 only at the N- or C-terminus or at both ends was carried out. To study the effect of hydrop- hobicity the residues ofpalmitic (Pal) and decanoic (Dec) acids were selected. Using a solid phase strategy we have synthesized unmodified peptide NDTKIAKK-Nle-A562-572-KA (1) and five of its acylated analogues, such as N[K(Dec)]DTKIAKK-Nle-A562-572-KA (2), NKDTKIAKK-Nle-A562-572-[K(Dec)]A (3), N[K(Dec)] DTKIAKK-Nle-A562-572-[K(Dec)]A (4), N[K(Pal)]DTKIAKK-Nle-A562-572-KA (5), and NKDTKIAKK-Nle- A562--572-[K(Pal)]A (6). Peptide 6 modified with palmitate at the C-terminus to a large extent increased the ba- sal AC activity and reduced AC stimulating effect of human chorionic gonadotropin (hCG) in the testes of rats, peptides 3 and 4 modified with decanoate at the C-terminus were less effective, but exceeded in activity the un- modified peptide 1, while peptides 2 and 5 acylated at the N-terminus were little active. The action of peptides was characterized by the tissue and the receptor specificity. Thus, the modification of LHR-peptide 562-572 with fatty acid radicals at the C-terminus increases its regulatory effect on the functional activity of the adenyla- te cyclase system in the rat testes, indicating promising the modification of GPCR-peptides with hydrophobic radicals. These data support the hypothesis that the hydrophobic radical to be localized in the locus of GPCR-peptide, where a transmembrane domain is located in the receptor.

Differential activation of soluble guanylate cyclase by a series of aryl disulfanyl dinitrate esters

Classic organic nitrates, such as nitroglycerin (GTN), are important vasodilators classed as NO donors, since biological activity is assumed to result entirely from bioactivation to NO and activation of soluble guanylate cyclase (sGC). Para-substituted aryl disulfanyl nitrate esters (SS-nitrates), designed based on a sulfhydryl-dependent mechanism of NO release, were investigated for their sGC activation. To obtain further insights into their interaction with hemoproteins, SS-nitrates were reacted with oxyhemoglobin (O2-Hb). SS-nitrates activated sGC with higher efficacy than GTN in the presence of cysteine. Some SS-nitrates, unlike GTN, activated sGC in the absence of cysteine. Linear correlations between the σp Hammett parameters and sGC activation efficacies or rates of O2-Hb oxidation by SS-nitrates in the presence of cysteine were found. Our results validate the design of SS-nitrates to circumvent the need for cysteine-mediated bioactivation and hint at NO-dependent and NO-independent mechanisms of sGC activation.

Potentiation of YC-1 activation of soluble guanylate cyclase by NO donors and the increase of the synergistic effect of YC-1 on the NO-dependent activation of the enzyme by 1,2,3-triazolyl-1,2,5-oxadiazole derivatives

Potentiation of YC-1 activation of soluble guanylate cyclase by NO donors and the increase of the synergistic effect of YC-1 on the NO-dependent activation of the enzyme by 1,2,3-triazolyl-1,2,5-oxadiazole derivatives

Adenylyl cyclase regulation in heart failure due to myocardial infarction in rats

Cardiac adenylyl cyclase (AC) activity was described to be differentially regulated in left and right ventricles (LVs and RVs) of rats with heart failure (HF) due to LV myocardial infarction (MI) (Sethi et al. Am J Physiol 272:H884-H893, 1997). AC activities in LVs and RVs were increased and decreased respectively in rats 8 and 16weeks post MI under basal and stimulatory conditions including AC activation via β-adrenergic receptors (β-ARs), stimulatory G protein (Gs), and direct AC activation with forskolin (FS). The current study aimed to detect alterations in rat heart AC activities in a comparable model of HF 9weeks post LV MI. Therefore, cardiac AC activities were measured under basal and β-AR-, Gs-, or FS-stimulated conditions as well as under inhibition with various MANT [2'(3')-O-(N-methylanthraniloyl)]-nucleotide AC inhibitors and the P-site AC inhibitors NKY80 [2-amino-7-(2-furanyl)-7,8-dihydro-5(6H)-quinazolinone] and vidarabine (9-β-D-arabinosyladenine, AraAde). Basal and stimulated AC activities along with AC inhibition experiments did not reveal evidence for changes in AC activity in LVs and RVs from MI group animals despite the presence of congestive HF. However, our study is indeterminate. Further studies are required to identify the factors responsible for previously described changes in cardiac AC activity in MI induced HF and to elucidate the role of altered AC regulation in the pathophysiology of HF. In order to detect small changes in AC regulation, larger group sizes than the ones used in our present study are required.

Potentiation of NO-dependent activation of soluble guanylate cyclase by 5-nitroisatin and the antiviral agent arbidol

Isatin (indole-dione-2,3) is an endogenous indole that exhibits a wide spectrum of biological and pharmacological activities. The effect of isatin derivatives, 5-nitroisatin and arbidol (an antiviral agent) on spermine NONO-induced activation of human platelet soluble guanylate cyclase has been investigated. 5-Nitroisatin and arbidol had no effect on basal activity, but synergistically increased in a concentration-dependent manner the spermine NONO-induced activation of this enzyme. 5-Nitroisatin and arbidol, like YC-1, sensitized guanylate cyclase towards nitric oxide (NO) and produced a leftward shift of the spermine NONO concentration response curve. However, both compounds did not influence the activation of guanylate cyclase by YC-1 and did not change the synergistic increase of spermine NONO-induced activation of soluble guanylate cyclase in the presence of YC-1. This suggests that 5-nitroisanin and arbidol did not compete with YC-1. Addition of isatin did not change the synergistic increase in the spermine NONO-induced guanylate cyclase activation by 5-nitroisatin and arbidol and did not influence a leftward shift of the spermine NONO concentration response curve produced by these compounds. These data suggest lack of competitive interaction between isatin and both its derivatives used.

Functional Analysis of c-di-GMP Pathway in Biomining Bacteria <i>Acidithiobacillus</i> <i>thiooxidans</i>

Bioleaching is the solubilization of metals catalyzed by acidophilic microorganisms. Among these, the Gram negative γ-proteobacteria from the genusAcidithiobacillusplay a relevant role. Biofilm formation has an impact on bioleaching performance. InAcidithiobacillussp. the molecular mechanisms involved in biofilm formation are still unknown. However, in many Gram-negative and some Gram-positive bacteria, the intracellular levels of the second messenger cyclic diguanilic acid (c-di-GMP) control the regulation of biofilm formation. These levels depend on the balance between the enzymatic activities of diguanylate cyclases (synthesis) and phosphodiesterases (degradation). Previously, functional c-di-GMP pathways have been characterized inAt.ferrooxidansandAt.caldus. Here, our main goal was to characterize the presence of a functional c-di-GMP pathway inAt.thiooxidans. By bioinformatic analysis, several putative-ORFs encoding DGCs, PDEs and effector proteins have been identified in the genome sequence ofAt.thiooxidansATCC 19377. RT-PCR experiments revealed that most of these ORFs are transcribed. In addition, the analysis of nucleotide-enriched fraction extracted fromAt.thiooxidanscells showed that this bacterium is able to produce c-di-GMP. These results strongly indicate thatAt.thiooxidanshas a functional c-di-GMP pathway.

Genetic variants of ANP and cardiometabolic protection: from populations to novel therapeutics

Background The cardiac hormone atrial natriuretic peptide (ANP) induces natriuresis, vasodilation and inhibits aldosterone through the activation of the guanylyl cyclase A receptor (GC-A) and the second messenger cGMP. ANP possesses also metabolic properties enhancing lipolysis and release of the adipokine adiponectin. Previous studies in general populations reported that the minor G allele of the ANP genetic variant rs5068 is associated with increased circulating levels of ANP and B-type natriuretic peptide, lower blood pressure (BP), and reduced risk of hypertension. We recently reported that in the general population from Olmsted County, MN, USA the G allele of rs5068 is associated not only with increased levels of ANP and lower BP but also with lower BMI, prevalence of obesity and metabolic syndrome. To advance our understanding of the phenotype associated with rs5068 we analyzed a community-based cohort from Sicily, Italy. Our second aim was to advance a potential therapeutics for cardiometabolic disease employing a novel long-acting ANP, MANP whose biological actions were defined in canines and in a rat model of metabolic syndrome.

G-protein signaling modulator 1 is a modifier of sGC-dependent vascular response

Background Earlier investigations demonstrated that the activity and function of soluble guanylyl cyclase (sGC) may be modulated by interacting proteins, such as HSP70, HSP90, CCTh or PDI. We have also previously demonstrated that G-protein signaling modulator proteins 1 and 2 (GPSM1, 2) directly interact with the catalytic region of sGC and attenuate the response of sGC to activators in cell lysates.

Thiol-redox proteins interact with soluble guanylyl cyclase and modulate its activity

Background Nitric oxide (NO) signalling through activation of soluble guanylyl cyclase (sGC) is key for the physiology of the cardiovascular and neuronal systems. Discrepancies in sGC activation and deactivation in vitro vs. in vivo have led to a search for endogenous factors that regulate sGC or assist in cellular localization. In our previous work, which identified Hsp70 as a modulator of sGC, we determined that protein disulfide isomerase (PDI) bound to an sGC-affinity matrix [1]. As several studies have reported that thiol oxidants and reductants modulate sGC activity, we sought to characterize the potential interaction between sGC and PDI, an enzyme that not only isomerases disulfide bonds but also regulates some proteins activity via interaction with its cysteine (Cys) active sites [2,3]. Results Using protein chemistry and purified sGC and PDI, we first showed that reduced sGC was modified by oxidized PDI in non-reducing Western blot and that sGC thiol titer was altered under these conditions. Moreover a PDI-Flag resin retains specifically sGC and this complex was resolved in the presence of a thiol-reductant. These data suggested that the mechanism of interaction between sGC and PDI is by mixed-disulfide exchange. In addition, PDI modulated NO-stimulated sGC activity while a PDI mutated in its two Cys active sites did not. A relatively novel method, in situ PLA (proximity ligation assay) established that endogenous PDI and sGC also interact in the CRL 2018 smooth muscle cells suggesting that the interaction between sGC and PDI is biologically relevant. Notably, both a 1a ndb 1s GC subunits of sGC showed potential co-localization with PDI [4]. Discussion These results underlined the significance of redox-thiol modulation of sGC and support past and current research showing that certain sGC Cys are necessary for such functions as enzyme activation, dimerization, and desensitization by S-nitrosation. A novel Mass Spectrometry analysis is currently applied to identify sGC Cys disulfide bonds and combined with mutational analysis and molecular dynamics simulation should unveil their involvement in interaction with PDI and their relevance as a new mechanism of sGC modulation.

Chronic activation of heme free soluble guanylate cyclase leads to cardio-renal protection in experimental hypertension

Background The cytoprotective NO/sGC/cGMP-signalling pathway is impaired under oxidative stress conditions as observed for cardiovascular diseases due to oxidation and subsequent loss of the prosthetic sGC heme group [1,2]. Thus, the pool of heme free sGC is increased under pathological conditions. The unique property of sGC activators such as cinaciguat (BAY 58—2667) to selectively activate heme free sGC allows to target the disease associated form of sGC. In this study a therapeutic effect of long-term activation of heme free sGC was investigated in an experimental model of salt sensitive hypertension.

Discovery of neuroprotective soluble guanylate cyclase modulators (GCMs) aciting via NO/GC/cGMP/CREB pathway

NO/cGMP signaling is essential for normal brain function, including learning and memory, and mediation of long-term potentiation (LTP). NO/cGMP signaling is coupled to cholinergic, glutamatergic, and dopaminergic systems and plays key roles in motor function associated with Parkinson’s disease pathogenesis and L-DOPA therapy. In Alzheimer’s Disease (AD), early synaptic failure, has been linked to dysfunction of gene expression programs mediated via the transcription factor cAMP-response element binding protein (CREB), activation of which is tightly regulated by NO/cGMP. Since several neurodegenerative disorders continue to be dogmatically linked to the chemical toxicity of NO, activation of soluble guanylyl cyclase (sGC) represents a potential therapeutic approach in both AD and PD; whereas negative modulation of sGC may be of use in some stages of PD. Such small molecule sGC modulators (GCMs) can be assayed in cell cultures by measuring levels of phosphorylated CREB (pCREB). GCMs that allosterically potentiate NO activation of sGC have proven successful in clinical trials for peripheral indications, however, there are no reports directed at therapeutic activity in the CNS. The aim of this study is to develop GCMs for use in CNS disorders, including AD and PD. A library of GCMs was synthesized, including positive allosteric modulators (PAMs) and negative modulators, as assessed by increasing levels of pCREB in SH-SY5Y human neuroblastoma cell cultures. Molecules were designed by classical bioisosteric replacement, aiming for desirable physiochemical properties and chemical diversity. GCM PAMs were assayed for cGMP elevation and reversal of neurotoxicity induced by 6-hydroxydopamine in dopaminergic neuronal cells. One GCM PAM, active in cell culture, was further tested for the reversal of memory deficits in mice treated with scopolamine, and drug levels in brain and plasma measured. The ligand binding site for sGC stimulators and GCM PAMs on the sGC protein remains to be definitively defined. Photoaffinity probes were designed that retained the activity of the parent GCM PAM in cell cultures in order to elucidate the binding mode to sGC and to indicate other protein partners for such compounds.

Soluble guanylyl cyclase as a therapeutic target in chronic obstructive pulmonary disease (COPD)

Background Resistive breathing (RB) due to airflow limitation is the pathophysiologic hallmark of chronic obstructive pulmonary disease (COPD). Nitric oxide (NO) is a physiological regulator of smooth muscle tone that acts through activation of soluble guanylyl cyclase (sGC). We hypothesized that increased smooth muscle tone limiting airflow in COPD could result from reduced sGC. Herein, we investigated the expression and downstream signalling of sGC in RB.

M-atrial natriuretic peptide and nitroglycerin in experimental acute hypertensive heart failure: differential actions of two cGMP activating therapeutics

Background Systemic hypertension is a common characteristic in acute heart failure (HF). This increasingly common phenotype is frequently associated with renal dysfunction and requires renal enhancing therapies. In a canine model of HF and acute hypertension we defined the cardiorenal actions of M-atrial natriuretic peptide (MANP), a novel particulate guanylyl cyclase A receptor activator (pGC-A) developed at the Mayo Clinic, and nitroglycerin (NTG), a soluble guanylyl cyclase (sGC) activator. Compared to native ANP, MANP has more sustained renal enhancing, blood pressure lowering and aldosterone inhibiting action. Based on the expression of the pGC-A receptor in the glomerulus, renal tubule and adrenal gland, compared to the more widespread expression of sGC in the renal vasculature, we hypothesized that MANP would have a more robust effect upon the glomerular filtration rate (GFR) and natriuretic actions and with greater aldosterone suppressing effects than sGC activation with NTG. To test these hypotheses, we used a canine model of combined HF and hypertension. Specifically, HF was induced by chronic (10 days) rapid right ventricular pacing (180 bmp), while hypertension was induced by continuous angiotensin II infusion (on day 11). We than characterized the cardiorenal and humoral actions of intravenous MANP (n=7), NTG (n=7), and vehicle (n=7) infusion. Results Mean arterial pressure (MAP) was reduced by both MANP (139±4 to 118±3 mmHg, p<0.05) and NTG (137 ±3 to 116±4 mmHg, p<0.05); similar findings were observed for reductions in pulmonary wedge pressure with MANP (12±2 to 6±2 mmHg, p<0.05) and NTG (12±1 to 6±1 mmHg, p<0.05). MANP enhanced renal function with significant increases in GFR (38±4 to 53±5 ml/min, p<0.05), renal blood flow (132±18 to 236±23 ml/ min, p<0.05), and natriuresis (11±4 to 689±37 mEq/min, p<0.05) with inhibition of aldosterone (32±3 to 23±2 ng/ dL, p<0.05), whereas NTG had no significant effect on these renal parameters or aldosterone activation.

NO/cGMP/pCREB re-activation reverses cognition deficits and attenuates amyloid-β neuropathology in transgenic models of Alzheimer’s disease

Results and Conclusion The hypothesis that activation of CREB through NO/ cGMP signaling might modify the amyloid-b (Ab) neuropathology, linked to AD pathogenesis, was demonstrated in both APP/PS1 and 3xTg transgenic mouse models of AD using small molecules, termed nomethiazoles, also designed to provide neuroprotection and attenuate pro-inflammatory cytokine release. Functional restoration of long-term potentiation was shown in hippocampal slices from AD transgenic mice in accord with observation of restoration of cognitive function in vivo, and was dependent upon soluble guanylyl cyclase (sGC) activation. Levels of pCREB and BDNF were significantly elevated, whereas TNFa, Ab, oligomeric Ab142, and also tau protein were significantly lowered after drug treatment. In the absence of neuronal loss in animal models of AD, neuroprotection was demonstrated in rat primary neurons after oxygen-glucose deprivation or application of oligomeric Ab. The lead nomethiazole was also studied in a novel transgenic mouse model, E4FAD, which incorporates familial AD mutations, but also has targeted replacement of mouse apolipoproteinE with human ApoE4, the major genetic risk factor for sporadic and age-related AD.

The Role of Heat Shock Protein 70 in the Protective Effect of YC-1 on β-Amyloid-Induced Toxicity in Differentiated PC12 Cells

Neurodegenerative brain disorders such as Alzheimer’s disease (AD) have been well investigated. However, significant methods for the treatment of the progression of AD are unavailable currently. Heat shock protein 70 (Hsp70) plays important roles in neural protection from stress by assisting cellular protein folding. In this study, we investigated the effect and the molecular mechanism of YC-1, an activator of guanylyl cyclase (GC), on Aβ25–35-induced cytotoxicity in differentiated PC12 cells. The results of this study showed that Aβ25–35 (10 µM) significantly increased p25 protein production in a pattern that was consistent with the increase in μ-calpain expression. Moreover, Aβ25–35 significantly increased tau hyperphosphorylation and induced differentiated PC12 cell death. YC-1 (0.5–10 µM) prevented the cell death induced by Aβ25–35. In addition, YC-1 (1, 10 µM) significantly blocked Aβ25–35-induced μ-calpain expression and decreased the formation of p25 and tau hyperphosphorylation. Moreover, YC-1 (5–20 µM) alone or combined with Aβ25–35 (10 µM) significantly increased the expression of Hsp70 in differentiated PC12 cells. The neuroprotective effect of YC-1 was significantly attenuated by an Hsp70 inhibitor (quercetin, 50 µM) or in PC12 cells transfected with an Hsp70 small interfering RNA. However, pretreatment of cells with the GC inhibitor ODQ (10 µM) did not affect the neuroprotective effect of YC-1 against Aβ25–35 in differentiated PC12 cells. These results suggest that the neuroprotective effect of YC-1 against Aβ25–35-induced toxicity is mainly mediated by the induction of Hsp70. Thus, YC-1 is a potential agent against AD.

Intracellular NAADP increase induced by extracellular NAADP via the P2Y11-like receptor

The aim of the study was to identify a signalling pathway allowing NAADP-induced intracellular NAADP increase and involving the P2Y11-like receptor. P2Y11-like and β-adrenergic receptors may play important regulatory roles within the cardiovascular system. Both receptors have been shown to be involved in triggering myocardial preconditioning. Using a Langendorff model we report a positive inotropic response induced by extracellular NAADP via P2Y11-like receptor stimulation. In cardiomyocyte cultures, P2Y11-like receptor stimulation by extracellular NAADP ([NAADP]e) increased intracellular cADP-ribose and NAADP concentration as evidenced by direct measurements. NF546, a new selective P2Y11 receptor agonist, increased intracellular cAMP, cADP-ribose and NAADP concentration confirming the involvement of the P2Y11-like receptor in this signalling pathway. NF157, a P2Y11 receptor antagonist, suppressed the increase in intracellular cADPr, NAADP and NAAD induced by either [NAADP]e or NF546. The response profile for intracellular cADP-ribose and NAADP concentration following P2Y11-like stimulation with NF546 was similar to reported data relating β-adrenergic stimulation with isoprenaline. This response represents the signature of the Gs/ADP-ribosyl cyclase activity. Moreover, this study provides a signalling pathway: intracellular NAADP increase induced by extracellular NAADP via metabotropic activity of P2Y11-like receptor. This pathway implying P2Y11-like could take part in the intracellular calcium rise reported for extracellular NAADP.

Investigation of the role of the NO–cGMP pathway on YC-1 and DEA/NO effects on thoracic aorta smooth muscle responses in a rat preeclampsia model

The present study was designed to investigate the effects of YC-1, a nitric oxide (NO)-independent soluble guanylate cyclase (sGC) activator, and DEA/NO, a NO donor, on smooth muscle responses in the preeclampsia model with suramin-treated rats and on the levels of cyclic guanosine monophosphate (cGMP) of thoracic aorta rings isolated from term-pregnant rats. Rats of 2 groups, control group and suramin group, were given intraperitoneal injection of saline or suramin, respectively. Suramin injection caused increased blood pressure, protein in urine, and fetal growth retardation. Thoracic aorta rings were exposed to contractile and relaxant agents. KCl contraction and papaverine relaxation responses were similar. Relaxation responses of YC-1 and DEA/NO decreased in suramin group. In both groups in the presence of ODQ, a sGC inhibitor, the relaxation responses of YC-1 and DEA/NO decreased. The cGMP content was determined by radioimmunoassay technique. The content of cGMP in the suramin group decreased. In the presence of YC-1 and DEA/NO in both groups, cGMP content increased, but in ODQ-added groups, there was a significant decrease. We conclude that in preeclampsia, the decrease of relaxation responses and the decrease of cGMP content could be due to the reduction in stimulation of sGC and the decrease in cGMP levels.

Functional activity of adenylyl and guanylyl cyclases in human spermatozoa with different motility

The most important functional characteristics of ejaculated spermatozoa is their ability to directional sustained movement that to a large extent determines their fertility. It is assumed that the enzymes with cyclase activity--adenylyl cyclase (AC) and guanylyl cyclase (GC), soluble and membrane-bound forms of which are found in human and mammalian sperm, play a key role in the regulation of motility. However, the functional activity of the cyclases in ejaculated spermatozoa with different motility and their contribution to the regulation of this process is virtually unexplored. The aim of this work was the functional characteristics of AC and GC in human sperm ejaculates with different content of motile forms of spermatozoa, and the study of regulation of these enzymes by hormones and non-hormonal agents. We found the differences in the activity and regulatory properties of AC and GC in the ejaculates differing in the content of motile forms of sperm. The basal AC activity and its sensitivity to bicarbonate anion and manganese cations, activators of cytosolic AC (cAC), were increased in ejaculates with a high proportion of motile spermatozoa. At the same time, AC effect of forskolin, GppNHp and adrenergic receptor agonists acting via membrane-bound AC (mAC) were significantly reduced in this case. Cytosolic GC in the ejaculates with a high proportion of motile spermatozoa was more sensitive to manganese cations, but the basal activity of GCs was altered slightly. The increase in the content of motile spermatozoa in the ejaculates led to decrease in the sensitivity of receptor GC to CNP, while the sensitivity to ANP was maintained, which indicated a change in the pattern of enzyme regulation with natriuretic peptides in favor of ANP, an important regulator of sperm chemotaxis. Thus, we have concluded that the change in the proportion of motile spermatozoa in the ejaculate induces changes in the functional activity and regulatory properties of soluble and membrane-bound forms of AC and GC, which can be used to control the motility, chemotaxis, acrosomal reaction and other processes determining the fertility of male germ cells.

BAY 60–2770 activation of soluble guanylate cyclase during ischemia protects against reperfusion injury

Previous studies showed that soluble Guanylate Cyclase (sGC) activation protects against postischemic reperfusion inflammation when used as a preconditioning compound in wild type (WT) H129, heme‐oxygenase‐1 KO (HO‐1 −/−) and eNOS −/−mice. We hypothesized that the same protective effect would occur when administered near the end of an ischemic episode. Intravital fluorescence microscopy was used to visualize leukocyte rolling (LR) and adhesion (LA) in single postcapillary venules of the small intestine. Circulating neutrophil counts were done using a hemo‐cytometer while TNF (Tumor Necrosis Factor)‐α was measured by an enzyme‐linked immunosorbent assay. The intra venous administration (30μg/kg) of BAY 60–2770 (sGC activator) during the last 5 min of the 45 min ischemic episode caused the postischemic LR and LA to remain at sham levels in the three strains of mice. Increases in intestinal TNF‐α and circulating neutrophils were also prevented. These results indicate that treatment with a sGC activator before initiating reperfusion can convey protection from an inflammatory response in WT, in mice with an impaired antioxidant system, and in the absence of expressed eNOS. This may provide an important strategy to minimize the damage done by and during treatment of occlusive vascular disease. Supported by: P01‐HL 095486 &amp; R01‐AA14945. BAY 60–2770 was a gift from Bayer Schering Pharma AG.

Endothelin-1 nociceptive signaling in keratinocytes may involve sensitization of adenylate cyclase

Endothelin-1 (ET-1) is an endogenous peptide implicated in pain from skin incision, inflammation, neuropathy, and cancer. ET-1 binds to GPCRs ETA and ETB onmany different cell types, includingneurons, endothelial cells, and keratinocytes. ET-1 injected into the skin is thought to cause nociception via a twophase mechanism — an early phase involving keratinocytes and a late phase involving ETA -mediated opening of TRPV1 and Na + channels in dorsal root ganglion neurons. Previouslywe reported that ET-1 elicits a rise in [Ca]i in proliferating human keratinocytes via ETA. We extend these findings by showing ET-1 modulation of the cAMP pathway in keratinocytes. We show that in keratinocytes, direct activation of adenylate cyclase by forskolin is potentiated by ET-1 (as measured by [cAMP]i accumulation). Using fluorescent microscopy Ca imaging via the ratiometric dye Fura-2, we further show that indirect activation of adenylate cyclase by the non-selective b-adrenergic agonist isoproterenol is also potentiated by ET-1 (as measured by transient [Ca]i increase) and this potentiation is abrogated by ETA but not ETB antagonists. Finally, we show that ET-1 does not directly activate endogenous adenylate cyclases (as measured by [cAMP]i accumulation). Thus, in keratinocytes ET-1 acts via ETA on endogenous adenylate cyclases to potentiate the [cAMP]i response to forskolin as well as the [Ca]i response to isoproterenol. Since ET-1 does not promote [cAMP]i accumulation directly, this phenomenon likely reflects Ca sensitization of endogenous adenylate cyclases. Further elucidation of ET-1 signaling pathways in keratinocytes could provide targets for pharmacological intervention that would ideally attenuate cutaneous pain mediated by ET-1.