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The hypocretin/orexin system regulates arousal through central nervous system mechanisms and plays an important role in sleep, wakefulness and energy homeostasis. It is unclear whether hypocretin peptides are also present in blood due to difficulties in measuring reliable and reproducible levels of the peptides in blood samples. Lack of hypocretin signalling causes the sleep disorder narcolepsy type 1, and low concentration of cerebrospinal fluid hypocretin-1/orexin-A peptide is a hallmark of the disease. This measurement has high diagnostic value, but performing a lumbar puncture is not without discomfort and possible complications for the patient. A blood-based test to assess hypocretin-1 deficiency would therefore be of obvious benefit. We here demonstrate that heating plasma or serum samples to 65°C for 30 min at pH 8 significantly increases hypocretin-1 immunoreactivity enabling stable and reproducible measurement of hypocretin-1 in blood samples. Specificity of the signal was verified by high-performance liquid chromatography and by measuring blood samples from mice lacking hypocretin. Unspecific background signal in the assay was high. Using our method, we show that hypocretin-1 immunoreactivity in blood samples from narcolepsy type 1 patients does not differ from the levels detected in control samples. The data presented here suggest that hypocretin-1 is present in the blood stream in the low picograms per millilitres range and that peripheral hypocretin-1 concentrations are unchanged in narcolepsy type 1., Graphical Abstract Graphical Abstract, The hypocretin/orexin system in the brain regulates sleep and wakefulness. Aegidius et al. have developed a simple test to measure hypocretin-1 peptide in blood samples, and show that peripheral hypocretin-1 concentrations are unchanged in narcolepsy type 1 despite being absent in the brains of these patients.

Multiple myeloma is a malignant expansion of plasma cells and aggressively affects bone health. We show that P2X7 receptor altered myeloma growth, which affects primary bone cells in vitro. Expression on six human myeloma cell lines confirmed the heterogeneity associated with P2X7 receptor. Pharmacology with 2&prime, (3&prime, )-O-(4-benzoylbenzoyl) adenosine 5&prime, triphosphate (BzATP) as agonist showed dose-dependent membranal pores on RPMI-8226 (p = 0.0027) and blockade with P2X7 receptor antagonists. Ca2+ influx with increasing doses of BzATP (p = 0.0040) was also inhibited with antagonists. Chronic P2X7 receptor activation reduced RPMI-8226 viability (p = 0.0208). No apoptosis or RPMI-8226 death was observed by annexin V/propidium iodide (PI) labeling and caspase-3 cleavage, respectively. However, bromodeoxyuridine (BrdU) labelling showed an accumulation of RPMI-8226 in the S phase of cell cycle progression (61.5%, p = 0.0114) with significant decline in G0/G1 (5.2%, p = 0.0086) and G2/M (23.5%, p = 0.0015) phases. As myeloma pathology depends on a positive and proximal interaction with bone, we show that P2X7 receptor on RPMI-8226 inhibited the myeloma-induced suppression on mineralization (p = 0.0286) and reversed the excessive osteoclastic resorption. Our results demonstrate a view of how myeloma cell growth is halted by P2X7 receptor and the consequences on myeloma&ndash, osteoblast and myeloma&ndash, osteoclast interaction in vitro.

Glibenclamide inhibits sulfonylurea receptor (SUR), which regulates several ion channels including SUR1-transient receptor potential melastatin 4 (SUR1-TRPM4) channel and ATP-sensitive potassium (KATP) channel. Stroke upregulates SURl-TRPM4 channel, which causes a rapid edema formation and brain swelling. Glibenclamide may antagonize the formation of cerebral edema during stroke. Preclinical studies showed that glibenclamide inhibits KATP channel-induced vasodilation without altering the basal vascular tone. The in vivo human cerebrovascular effects of glibenclamide have not previously been investigated.In a randomized, double-blind, placebo-controlled, three-way cross-over study, we used advanced 3 T MRI methods to investigate the effects of glibenclamide and KATP channel opener levcromakalim on mean global cerebral blood flow (CBF) and intra- and extracranial artery circumferences in 15 healthy volunteers. Glibenclamide administration did not alter the mean global CBF and the basal vascular tone. Following levcromakalim infusion, we observed a 14% increase of the mean global CBF and an 8% increase of middle cerebral artery (MCA) circumference, and glibenclamide did not attenuate levcromakalim-induced vascular changes. Collectively, the findings demonstrate the vital role of KATP channels in cerebrovascular hemodynamic and indicate that glibenclamide does not inhibit the protective effects of KATP channel activation during hypoxia and ischemia-induced brain injury.

Background and Purpose Endothelins act via two receptor subtypes, ETA and ETB. Under physiological conditions in coronary arteries, ETA receptors expressed in smooth muscle cells mediate vasoconstriction whereas ETB receptors mainly found in endothelial cells mediate vasorelaxation. However, under pathophysiological conditions, ETB receptors may also be expressed in vascular smooth muscle cells mediating vasoconstriction. Here, we have investigated whether vasoconstrictor ETB receptors are up-regulated in coronary arteries after experimental myocardial ischaemia in rats. Experimental Approach Male Sprague-Dawley rats were subjected to either heart ischaemia–reperfusion (15 min ischaemia and 22 h reperfusion), permanent ischaemia (22 h) by ligation of the left anterior descending coronary artery, or sham operation. Using wire myography, the endothelin receptor subtypes mediating vasoconstriction were examined in isolated segments of the left anterior descending and the non-ligated septal coronary arteries. Endothelin receptor-mediated vasoconstriction was examined with cumulative administration of sarafotoxin 6c (ETB receptor agonist) and endothelin-1 (with or without ETA or ETB receptor blockade). The distribution of ETB receptors was localized with immunohistochemistry and quantified by Western blot. Key Results Endothelin ETB receptor-mediated vasoconstriction and receptor protein levels were significantly augmented in coronary arteries situated downstream of the occlusion after ischaemia–reperfusion compared with non-ischaemic arteries. In contrast, the ETA receptor-mediated vasoconstriction was unaltered in all groups. Conclusions and Implications Ischaemia–reperfusion induced local up-regulation of ETB receptors in the smooth muscle cells of coronary arteries in the post-ischaemic area. In contrast, in non-ischaemic areas, ETB receptor function was unaltered.

INTRODUCTION: Cigarette smoking is a well-known risk factor for developing cardiovascular diseases, but the underlying mechanisms are largely unknown. Recent data suggest that vasocontractile receptor modulation could be an important factor. Surfactant protein D (SP-D) is important in the particle clearance in the lungs and knock-out (KO) mice for this protein develop emphysema. SP-D is also weakly expressed in the vasculature. We aimed to investigate whether SP-D was important in the cardiovascular response to cigarette smoke exposure (CSE), by utilizing SP-D KO mice and a myograph setup.METHODS: Wild type (WT) and SP-D KO mice were exposed to cigarette smoke (CS) or room air for 12 weeks. The pulmonary artery, left anterior descending coronary artery, and basilar artery (BA) were isolated and mounted in wire myographs. Contractile concentration response curves to endothelin-1 and UDP were obtained.RESULTS: CSE caused a leftward shift in the concentration response curves for endothelin-1 in the BA for both WT and SP-D KO. UDP, acting on the purinergic P2Y6 receptor, caused reduced contraction in the left descending artery and increased contraction in the BA in the CSE WT mice. SP-D KO mice displayed no smoke induced changes, but were surprisingly similar to the CSE WT.CONCLUSION: The contractility to UDP was altered in the brain and heart vasculature of CSE mice. SP-D KO (both control and CSE) and CSE WT had similar changes in contractility compared to control WT.IMPLICATIONS: These results show that sub-chronic smoking induces vascular changes in the WT, mainly for the purinergic P2Y6 receptor together with minor changes for the endothelin-1 receptor. SP-D KO (both control and CSE) does not show any further changes compared to CSE WT.

Activation of the trigeminovascular pain signalling system, including cerebral arteries, meninges, trigeminal ganglion, and brain stem, is involved in migraine. Furthermore, stimulation of cyclic nucleotide (cAMP and cGMP) production as well as inhibition of phosphodiesterases (PDEs) induces headache and migraine. In order to investigate the possible role of PDE in the pain pathway of migraine, expression of the most recently discovered PDE subtypes (9A, 10A and 11A) in cerebral arteries, dura mater, and trigeminal ganglion and nucleus was examined. The presence of mRNA and protein in the middle cerebral artery, basilar artery, meninges, trigeminal ganglion, and spinal trigeminal nucleus of male Sprague-Dawley rats were investigated using real-time PCR, Western blot, and immunohistochemistry. The results were compared to two peripheral arteries: aorta and mesenteric artery, as well as neocortex and cerebellar cortex. Real-time PCR and Western blotting showed that PDE9A, PDE10A and PDE11A are expressed in components of the rat trigeminovascular pain signalling system including middle cerebral artery, basilar artery, meninges, trigeminal ganglion and spinal trigeminal nucleus. Aorta and mesenteric artery as well as cerebral neocortex and cerebellar cortex also showed expression of PDE9A, PDE10A and PDE11A. Immunohistochemistry revealed that PDE9A, PDE10A and PDE11A are localised in the cytosol of nerve cell bodies of the trigeminal ganglion. We here present, for the first time, the expression of PDE9A, PDE10A, and PDE11A in the trigeminovascular system. The functional implications are yet unknown, but their localisation indicates that they may have a role in the pain pathway of migraine as well as trigeminal neuralgia and trigeminal autonomic cephalalgias.

Activation of the trigeminovascular pain signalling system appears involved in migraine pathophysiology. However, the molecular mechanisms are only partially known. Stimulation of cAMP and cGMP production as well as inhibition of their breakdown induce migraine-like headache. Additionally, migraine may be associated with mutations in ion channels. The aim of the present study was to describe the expression of phosphodiesterase 3 (PDE3) and 5 (PDE5) and cyclic nucleotide-gated ion channels (CNG) in cerebral arteries, meninges, and the trigeminal ganglion. mRNA for PDE and CNG was determined in the rat middle cerebral artery, basilar artery, trigeminal ganglion, and dura mater using real-time PCR. PDE and CNG proteins were identified using Western blot. For comparison, rat aorta and mesenteric artery were analysed. PDE3A, PDE3B, and PDE5A mRNA were detected in all tissues examined except for PDE3A mRNA in dura mater and the trigeminal ganglion. PDE5A and PDE3A protein expression was present in both cerebral and peripheral arteries, whereas PDE3B protein was present only in the cerebral arteries. The CNGA4 and B1 subunit mRNAs were detected in cerebral arteries and CNGA2 also in the mesenteric artery. CNGA2 and A3 proteins were found in cerebral arteries and dura and CNGA1, CNGA2 and CNGA3 in the trigeminal ganglion. In conclusion, PDE3A, PDE3B, PDE5A, and five CNG subunits were expressed in several components of the trigeminovascular system of the rat. This suggests that modulation of cAMP and cGMP levels by PDE and activation of CNG may play a role in trigeminovascular pain signalling leading to migraine headache.

Background: Coronary artery remodelling and vasospasm is a complication of acute myocardial ischemia and reperfusion. The underlying mechanisms are complex, but the vasoconstrictor peptide endothelin-1 is suggested to have an important role. This study aimed to determine whether the expression of endothelin-1 and its receptors are regulated in the myocardium and in coronary arteries after experimental ischemia-reperfusion. Furthermore, we evaluated whether treatment with a specific MEK1/2 inhibitor, U0126, modified the expression and function of these proteins. Methods and findings: Sprague-Dawley rats were randomly divided into three groups: sham-operated, ischemiareperfusion with vehicle treatment and ischemia-reperfusion with U0126 treatment. Ischemia was induced by ligating the left anterior descending coronary artery for 30 minutes followed by reperfusion. U0126 was administered before ischemia and repeated 6 hours after start of reperfusion. The contractile properties of isolated coronary arteries to endothelin-1 and sarafotoxin 6c were evaluated using wire-myography. The gene expression of endothelin-1 and endothelin receptors were measured using qPCR. Distribution and localization of proteins (pERK1/2, prepro-endothelin-1, endothelin-1, and endothelin ETA and ETB receptors) were analysed by Western blot and immunohistochemistry. We found that pERK1/2 was significantly augmented in the ischemic area 3 hours after ischemia-reperfusion; this correlated with increased ETB receptor and ET-1 gene expressions in ischemic myocardium and in coronary arteries. ETB receptor-mediated vasoconstriction was observed to be increased in coronary arteries 24 hours after ischemia-reperfusion. Treatment with U0126 reduced pERK1/2, expression of ET-1 and ETB receptor, and ETB receptor-mediated vasoconstriction. Conclusions: These findings suggest that the MEK-ERK1/2 signaling pathway is important for regulating endothelin-1 and ETB receptors in myocardium and coronary arteries after ischemia-reperfusion in the ischemic region. Inhibition of the MEK-ERK1/2 pathway may provide a novel target for reducing ischemia-reperfusion damage in the heart.

The neuropathology leading to migraine pain has centered on an either a vascular or neuronal origin. Sildenafil, a specific inhibitor of phosphodiesterase 5 (PDE5), was the first compound to induce migraine-like headache in a human headache model without a concomitant artery dilatation.

BackgroundPhosphodiesterases (PDEs) cleave phosphodiester bonds in cyclic nucleotides and play diverse roles in cell biology. PDE5A is a cytoplasmic phosphodiesterase which specifically degrades cyclic guanosine monophosphate (cGMP), a cell signaling molecule that plays important roles in neuronal signaling and vascular smooth muscle contraction. Inhibition of PDE5A induces headache resembling migraine headaches.AimTo test the hypothesis that Ser102 and Ser104 in PDE5A and/or their phosphoserine derivatives 1) regulate the intracellular localization and/or activity of PDE5A, and 2) modulate the interaction between PDE5A and pharmaceutical reagents in clinical or pre-clinical use for migraine headaches and other types of vascular dysfunction.MethodsWild type PDE5A or PDE5A with substitution mutations (Ser102Ala, Ser104Ala or Ser102Ala/Ser104Ala) were overexpressed in SK-N-AS neuroblastoma cells as C-terminal fusions with green fluorescent protein. Transfected cells were treated with sildenafil, cilostazol, glyceryl trinitrate, calcitonin gene-related peptide (CGRP) or sumatriptan. PDE5A-GFP fusion proteins were localized in fixed cells by immunofluorescence and PDE activity was quantified in cell extracts by standard in vitro assay using [3H] cGMP.ResultsThe intracellular distribution of wild-type, single and double mutant PDE5A was similar and was not altered by exposure to sildenafil, cilostazol, glyceryl trinitrate, calcitonin gene-related peptide (CGRP) or sumatriptan. PDE5 activity was similar for wild type, Ser102Ala and Ser104Ala PDE5A, but activity of the Ser102Ala/Ser104Ala mutant was approximately two-fold higher than wild type. Double mutant Ser102Ala/Ser104Ala migrated as a single band on a native acrylamide gel, while wild-type and single mutant PDE5A migrated as a doublet.InterpretationSer102 and Ser104 may influence the conformational flexibility of PDE5A, which may in turn influence phosphorylation status, allosteric regulation by cGMP or other as yet unknown regulatory mechanisms for PDE5A. PDE5A activation could be important in reversal of migraine-like headache.

Background Understanding of the neuropathology leading to migraine pain has centered on either a vascular or neuronal origin. Sildenafil, a specific inhibitor of phosphodiesterase 5 (PDE5), induces migraine-like headache in a human headache model without concomitant artery dilation. The presence and activity of PDE3 and PDE5 is known in cerebral arteries. However, the presence in the neuronal part of the trigeminovascular pathway, i.e. the trigeminal ganglion and the possible co-localization with calcitonin gene-related peptide (CGRP), is not known. Methods Rat trigeminal ganglia were isolated and immunohistochemistry and in situ hybridization was applied. Evaluations of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) hydrolysis were performed using scintillation proximity assays. Results PDE3 and PDE5 were present and active in the trigeminal ganglia. A subset of PDE3- and PDE5-positive neurons contained CGRP. In contrast to cGMP, both sildenafil and cilostazol influenced cAMP hydrolysis. Interpretation Sildenafil may exert its effect on the neuronal part of the migraine pain pathway. In addition to the effects on cGMP signaling, sildenafil may indirectly affect cAMP signaling in the trigeminal ganglion. This result may suggest a common cAMP-related pathway for sildenafil, cilostazol, and CGRP in eliciting migraine pain.

Background Tumour necrosis factor-α (TNF-α) is a pleiotropic pro-inflammatory cytokine, which is rapidly upregulated in the brain after injury. TNF-α acts by binding to its receptors, TNF-R1 (p55) and TNF-R2 (p75), on the cell surface. The aim of this study was first to investigate if there is altered expression of TNF-α and TNF-α receptors in cerebral artery walls following global or focal ischemia, and after organ culture. Secondly, we asked if the expression was regulated via activation of the MEK-ERK1/2 pathway. Methods The hypothesis was tested in vivo after subarachnoid hemorrhage (SAH) and middle cerebral artery occlusion (MCAO), and in vitro by organ culture of isolated cerebral arteries. The localization and amount of TNF-α, TNF-α receptor 1 and 2 proteins were analysed by immunohistochemistry and western blot after 24 and 48 h of organ culture and at 48 h following SAH or MCAO. In addition, cerebral arteries were incubated for 24 or 48 h in the absence or presence of a B-Raf inhibitor (SB386023-b), a MEK- inhibitor (U0126) or an NF-κB inhibitor (IMD-0354), and protein expression evaluated. Results Immunohistochemistry revealed enhanced expression of TNF-α, TNF-R1 and TNF-R2 in the walls of cerebral arteries at 48 h after MCAO and SAH compared with control. Co-localization studies showed that TNF-α, TNF-R1 and TNF-R2 were primarily localized to the cell membrane and the cytoplasm of the smooth muscle cells (SMC). There was, in addition, some expression of TNF-R2 in the endothelial cells. Immunohistochemistry and western blot analysis showed that these proteins were upregulated after 24 and 48 h in culture, and this upregulation reached an apparent maximum at 48 h of organ culture. Treatment with U0126 significantly reduced the enhanced SMC expression of TNF-α, TNF-R1 and TNF-R2 immunoreactivities after 24 and 48 h of organ culture. The Raf and NF-κB inhibitors significantly reduced organ culture induced TNF-α expression while they had minor effects on the TNF-α receptors. Conclusion The present study shows that cerebral ischemia and organ culture induce expression of TNF-α and its receptors in the walls of cerebral arteries and that upregulation is transcriptionally regulated via the MEK/ERK pathway.

Phosphodiesterase 5 (PDE5) is associated with migraine pathophysiology, stroke recovery and vasospasm treatment. The potential vascular interplay of PDE5 inhibitors sildenafil, tadalafil and UK-114,542 was studied by intra- versus extra-luminal administration in rat middle cerebral arteries in vitro and on middle meningeal arteries in vivo. By Western blot PDE5 was detected in both cerebral and meningeal arteries, though with minor variations in band intensity between vascular beds. Rat middle cerebral artery diameter was investigated using pressurised arteriography, applying UK-114,542, sildenafil, and tadalafil intra- or extra-luminally. Effects on the dural middle meningeal artery were studied in the in vivo closed cranial window model. At high concentrations, abluminal sildenafil and UK-114,542, but not tadalafil, induced dilatation of the middle cerebral artery. Luminal application elicited a contraction of 4% (sildenafil, P=0.03) and 10% (tadalafil, P=0.02). In vivo, sildenafil, but not tadalafil, dose-dependently dilated middle meningeal artery concomitant to blood pressure reduction (1-3mg/kg);1mg/kg sildenafil inducing 60 ± 14% (P=0.04) and vehicle (DMSO) 13 ± 6% dilatation. In conclusion, PDE5 inhibitors applied luminally had minor contractile effect, whereas abluminal sildenafil induced middle cerebral artery dilatation above therapeutic levels. In vivo, sildenafil dilated middle meningeal artery concomitant with a reduction in blood pressure. Tadalafil had no dilatory effects. PDE5 inhibitors show differential vascular activity in cerebral arteries from healthy animals; arterial dilatation is seen primarily above therapeutic levels. Such findings support clinical studies showing no vasodilator effects of sildenafil on cerebral arteries in healthy subjects.

Phosphodiesterases (PDEs) are essential regulators of cyclic nucleotide signaling. Little is known of the distribution and function of the cyclic adenosine monophosphate (cAMP) hydrolyzing PDE8A family. Employing immunohistochemistry and Western blots this study maps the distribution of PDE8A in the brain of adult male Sprague-Dawley rats and in the trigeminal ganglion. PDE8A was confined to neuronal perikaryal cytoplasm and to processes extending from those perikarya. The neurons exhibiting PDE8A-immunoreactivity were widely distributed in the forebrain, brain stem, and cerebellum. Strongly immunoreactive neurons were located in the olfactory bulb, the septal area, zona incerta, and reticular nucleus of the thalamus. Less immunoreactivity was seen in the hippocampus and cerebral cortex. Intense staining was detected in both the substantia nigra and the sensory trigeminal nucleus. In cerebellum PDE8A immunoreactivity was located not only in the Purkinje cells, but also in the granular cells as well as the parallel fibres in the molecular layer. PDE8A immunoreactivity was represented in the epithelial lining of the choroids plexus, the dura mater, and the neurons of the trigeminal ganglion. The localization of the cAMP degrading PDE8A may indicate a role for PDE8A in cAMP signaling related to pain transmission, motor function, cognition and olfaction.

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

Compounds which increase cGMP levels are implicated in migraine pathophysiology as well as stroke recovery. Inhibitors of the cGMP degrading enzyme phosphodiesterase type 5 (PDE5) induce headache and migraine in humans, however surprisingly and unlike other migraine inducing drugs without measurable dilatation of cerebral arteries [1] or changes in hemodynamic response or excitability [2]. We aimed to investigate whether sildenafil and tadalafil induced dilatation of rat middle cerebral and meningeal arteries in vitro and in vivo.

Contains fulltext : 47822.pdf (Publisher’s version ) (Open Access) The 90-kDa ribosomal S6 kinases (RSK1-3) are important mediators of growth factor stimulation of cellular proliferation, survival, and differentiation and are activated via coordinated phosphorylation by ERK and 3-phosphoinositide-dependent protein kinase-1 (PDK1). Here we performed the functional characterization of a predicted new human RSK homologue, RSK4. We showed that RSK4 is a predominantly cytosolic protein with very low expression and several characteristics of the RSK family kinases, including the presence of two functional kinase domains and a C-terminal docking site for ERK. Surprisingly, however, in all cell types analyzed, endogenous RSK4 was maximally (constitutively) activated under serum-starved conditions where other RSKs are inactive due to their requirement for growth factor stimulation. Constitutive activation appeared to result from constitutive phosphorylation of Ser232, Ser372, and Ser389, and the low basal ERK activity in serum-starved cells appeared to be sufficient for induction of approximately 50% of the constitutive RSK4 activity. Finally experiments in mouse embryonic stem cells with targeted deletion of the PDK1 gene suggested that PDK1 was not required for phosphorylation of Ser232, a key regulatory site in the activation loop of the N-terminal kinase domain, that in other RSKs is phosphorylated by PDK1. The unusual regulation and growth factor-independent kinase activity indicate that RSK4 is functionally distinct from other RSKs and may help explain recent findings suggesting that RSK4 can participate in non-growth factor signaling as for instance p53-induced growth arrest.

BackgroundPhosphodiesterase 5 (PDE5) is associated with migrainepathophysiology, stroke recovery and vasospasm treat-ment [1,2]. We have shown previously that vasodilata-tion was not a prerequisite for migraine induction;sildenafil elicited migraine-like attacks in migrainepatients without measurable changes in intra- or extra-cerebral artery diameter. Further, sildenafil was found tonot affect neurovascular response or excitability [3].However, dural artery responses were not accounted forin the human studies and minor vascular changes offunctional importance may not have been detected.The potential vascular interplay of PDE5 inhibitors sil-denafil, tadalafil and UK-114,542 were studied by intra-versus extra-luminal administration in rat middle cere-bral arteries (MCA) in vitro and on middle meningealarteries (MMA) in vivo.AimTo examine a possible vascular site of action, if any, ofeach of sildenafil and tadalafil by investigating 1) theeffects of PDE5 inhibitors in vitro dilatation of the mid-dle cerebral artery (MCA) with controlled luminal orextra-luminal application of the drugs and 2) the in vivoeffects of intravenous PDE5 inhibitors on the middlemeningeal artery (MMA) dilatation in a closed cranialwindow model in rats.MethodsRat MCA diameter was investigated using pressurisedarteriography, applying UK-114,542, sildenafil, andtadalafil intra- or extra-luminally. Effects on MMA werestudied in the in vivo closed cranial window model.ResultsAt high concentrations, abluminal sildenafil and UK-114,542, but not tadalafil, induced dilatation. Luminalapplication elicited a contraction of 4 % (sildenafil, p =0.03) and 10 % (tadalafil, p = 0.02).In vivo, sildenafil,but not tadalafil, dose-dependently dilated MMA conco-mitant to blood pressure reduction (1-3 mg/kg);1 mg/kgsildenafil inducing 60 ± 14 % (p = 0.04) and vehicle(DMSO) 13 ± 6 % dilatation.ConclusionPDE5 inhibitors applied luminally had contractile effecton MCA. Abluminal sildenafil induced MCA dilatationabove therapeutic levels. In vivo, sildenafil dilated MMA.Tadalafil had no dilatory effects. PDE5 inhibitors showdifferential vascular activity in arteries, although clini-cally the potential for headache induction appears simi-lar. Such findings support clinical studies showing novasodilatory effects of sildenafil on cerebral arteries inhealthy subjects.

Background Activation of the trigeminovascular pain signalling system, which includes cerebral arteries, meninges, trigeminal ganglion, and brain stem, may be involved in migraine. Furthermore, stimulation of cyclic nucleotide (cAMP and cGMP) production as well as inhibition of phosphodiesterases (PDEs) induces headache and migraine [1-4]. The aim is to study the expression of the most recently discovered PDE subtypes (9A, 10A and 11A) in cerebral arteries, the dura mater, and the trigeminal ganglion and nucleus. This may give a clue to a role in pathogenesis of migraine.