Emelie Stenman

Cerebral Ischemia Upregulates Vascular Endothelin ETB Receptors in Rat

Background and Purpose— Elevated levels of endothelin-1 (ET-1) have been reported in cerebral ischemia. A role for ET may prove more important if the vascular receptors were changed. We addressed whether there is any change in ET receptor expression in cerebral ischemia. Methods— The right middle cerebral artery (MCA) was occluded in male Wistar rats for 2 hours with the intraluminal filament method. The basilar artery and both MCAs were removed after 46 hours of recirculation. The contractile responses to ET-1, a combined ETA and ETB receptor agonist, and sarafotoxin 6c (S6c), a selective ETB receptor agonist, were examined in vitro, and ET receptor mRNA was quantified by real-time polymerase chain reaction. Results— S6c, which had no contractile effect per se on fresh or sham-operated rat cerebral arteries, induced a marked contraction in the occluded MCA (Emax [maximum contraction, calculated as percentage of the contractile capacity of 63.5 mmol/L K+]=68±68%;P <0.0001), while there was no difference in the responses to ET-1 after cerebral ischemia. Real-time polymerase chain reaction revealed a significant upregulation of both the ETA and ETB receptors (both P <0.05) in the occluded MCA compared with the nonoccluded MCA from the same rats. Conclusions— Focal cerebral ischemia in rat induces increased transcription of both ETA and ETB receptors, which results in the appearance of a contractile response to the ETB receptor agonist S6c. These results suggest a role for ET receptors in the pathogenesis of a vascular component after cerebral ischemia.

Cerebral Ischemia Enhances Vascular Angiotensin AT1 Receptor–Mediated Contraction in Rats

Background and Purpose— The aim of the study was to examine how focal cerebral ischemia affects the expression and function of vascular angiotensin II receptors. Materials and Methods— We used an intraluminal filament occlusion technique to occlude the right middle cerebral artery (MCA) of the rat. Myographs were used for functional studies of the MCA and real-time polymerase chain reaction, for determination of relative mRNA levels. Results— The contractile responses to angiotensin II were stronger in the right occluded MCA compared with the left MCA and the MCA from sham-operated rats 48 hours after MCA occlusion (P <0.05). The angiotensin II type 1 (AT1) receptor antagonists candesartan and losartan abolished the enhanced responses to angiotensin II (P <0.05), whereas the AT2 receptor antagonist PD123319 had no effect. The amount of AT1 receptor mRNA was lower in the occluded MCAs compared with nonoccluded MCAs 48 hours after occlusion (P <0.05), whereas the mRNA levels of angiotensin converting enzyme (ACE) were higher in the occluded arteries. The mRNA levels of the AT2 receptor and nuclear factor-&kgr;B were unchanged. Conclusions— Focal cerebral ischemia in the rat upregulated the contractile responses to angiotensin II in the ipsilateral MCA, and this contraction was mediated by AT1 receptors. Real-time polymerase chain reaction revealed decreased AT1 receptor mRNA levels in the occluded MCA, whereas the amount of ACE mRNA was increased, suggesting locally enhanced angiotensin II production. These results support a role for AT1 receptors in cerebral ischemia, and we think that AT1 receptors might be a future therapeutic target in ischemic stroke.

Intracellular Pathways Involved in Upregulation of Vascular Endothelin Type B Receptors in Cerebral Arteries of the Rat

Background and Purpose— Previous studies have shown that contractile endothelin type B (ETB) receptors are upregulated in cerebral arteries after experimental focal cerebral ischemia. The aim of this study was to examine the upregulation of contractile ETB receptors in cerebral arteries after organ culture and to elucidate the intracellular pathways involved. Methods— Rat middle cerebral arteries (MCAs) were incubated with or without inhibitors. The vessels were mounted in myographs, and the contractile responses to endothelin-1 (ET-1) (ETA and ETB receptor agonist) and sarafotoxin 6c (ETB receptor agonist) were measured. Levels of ETB receptor mRNA were measured with real-time polymerase chain reaction. Results— In fresh MCA, sarafotoxin 6c had no contractile effect. However, after organ culture, a strong concentration-dependent contraction was induced. ET-1 produced a strong contraction, in which the Emax was unaffected by organ culture but the EC50 was decreased with time. The sarafotoxin 6c–induced contraction after 24 hours of organ culture was attenuated by the transcriptional inhibitor actinomycin D and the translational inhibitor cycloheximide as well as the protein kinase C inhibitor Ro-31-8220. Real-time polymerase chain reaction revealed that the mRNA levels of the ETB receptor were increased after organ culture compared with fresh vessels. Actinomycin D and Ro-31-8220 diminished the enhanced mRNA levels considerably. Conclusions— The results suggest that, in fresh MCA, the ETA receptor is the most prominent subtype, while after organ culture ETB receptors also contribute to the contraction. This upregulation is due to de novo transcription of receptors. Protein kinase C is involved in the upregulation as Ro-31-8220 attenuates the contraction and the mRNA increase.

Cerebral ischemia induces transcription of inflammatory and extracellular-matrix-related genes in rat cerebral arteries

Cerebral ischemia results in a local inflammatory response that contributes to the size of the lesion, however, the involvement of the cerebral vasculature is unknown. We hypothesise that the expression of inflammatory genes (Il6, iNOS, cxcl2, TNF-α and Il-1β) and extracellular-matrix-related genes (MMP9, MMP13) is induced in cerebral arteries following cerebral ischemia via activation of mitogen activated kinases (MAPKs). This hypothesis was tested in vivo by experimental subarachnoid haemorrhage (SAH) and temporal middle cerebral artery occlusion (MCAO), and by organ culture of isolated cerebral arteries with quantitative real time PCR (mRNA expression) and immunohistochemistry (localization of protein expression). The gene promoters were investigated in silica with computer analysis. The mRNA analysis revealed that the ischemic models, SAH and MCAO, as well as organ culture of isolated cerebral arteries resulted in transcriptional upregulation of the abovementioned genes. The protein expression involved phosphorylation of three different MAPKs signalling pathways (p38, ERK 1/2 and SAPK/JNK) and the downstream transcription factors (ATF-2, Elk-1, c-Jun) shown by immunohistochemistry and quantified by image analysis. All three models revealed the same pattern of activation in the cerebrovascular smooth muscle cells. The in silica analysis demonstrated binding sites for said transcription factors. The results suggest that cerebral ischemia and organ culture induce activation of p38, ERK 1/2 and SAPK/JNK in cerebral arteries which in turn activate the transcription factors ATF-2, Elk-1 and c-Jun and the expression of inflammatory and extracellular-matrix-related genes in the wall of cerebral arteries.

Effects of tramadol on rat detrusor overactivity induced by experimental cerebral infarction

OBJECTIVE Cerebrovascular disease, such as stroke, frequently results in incontinence by reducing suprapontine micturition control. Intraluminal occlusion of the middle cerebral artery (MCA), which produces detrusor overactivity, has been introduced as a useful model of stroke-induced lower urinary tract dysfunction. Recently, the effective analgesic tramadol, was found to possess inhibitory actions on normal rat micturition. The current study aimed to examine the potential effect of tramadol on rat detrusor overactivity due to cerebral infarction. METHODS In female Sprague-Dawley rats, cerebral ischemia was induced by occlusion of the MCA and the urinary bladder was catheterised. Three days later, continuous cystometry was performed in awake animals and the effects of tramadol given intravenously were studied. RESULTS In cerebral infarcted rats, bladder capacity was lower (48+/-9%) and micturition pressure higher (76+/-21%) than in control rats. Tramadol 5 mg x kg(-1) given i.v., increased bladder capacity (59+/-29%) and threshold pressure (47+/-32%) to values similar to those in control rats. However, micturition pressure was not significantly altered. Tramadol induced diuresis in some, but not all, cerebral infarcted rats. CONCLUSION Tramadol normalised detrusor overactivity in MCA-occluded rats. The drug might have a treatment potential in patients with detrusor overactivity after stroke.

Reduction of bFGF-induced smooth muscle cell proliferation and endothelin receptor mRNA expression by mevastatin and atorvastatin.

The anti-atherosclerosis mechanisms of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) occur via both cholesterol-dependent and cholesterol-independent mechanisms. The present study used aortic and cerebral vascular smooth muscle cells (SMC) from rat to investigate whether atorvastatin and mevastatin affect basic fibroblast growth factor (bFGF)-induced SMC proliferation and the mRNA expression of endothelin A (ET(A)) and endothelin B (ET(B)) receptors. Cell proliferation was assessed by MTT and real-time PCR was used to quantify ET(A) and ET(B) receptor mRNA. bFGF-induced concentration and time dependent SMC proliferation and up-regulation of the mRNA expression of ET(A) and ET(B) receptors. The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors inhibited bFGF-induced proliferation of SMC (P<0.01). In aortic SMC atorvastatin and mevastatin significantly inhibited bFGF-induced mRNA expression of endothelin ET(A) and ET(B) receptors (P<0.05). Although in cerebral SMC the inhibitory effect of the statins was comparable in size with that seen in aortic SMC, only reached borderline significance (P=0.06) for ET(A) receptor mRNA but not for ET(B). The findings suggested a direct effect of statins on the vascular wall beyond their well-known lipid lowering effect in anti-atherosclerosis. Furthermore, the specific antagonists of ET(A) and ET(B) receptors (FR139317 and BQ788, respectively) significantly inhibited bFGF-induced SMC proliferation (P<0.001). The results suggested that endothelin receptors and the mevalonate pathway were involved in bFGF-induced SMC proliferation.

Cytokines and growth factors modify the upregulation of contractile endothelin ET(A) and ET(B) receptors in rat cerebral arteries after organ culture.

Experimental cerebral ischaemia and organ culture of cerebral arteries induce an increased endothelin ETB receptor–mediated contraction. The aim of this study was to examine whether cytokines and growth factors, known to be activated in ischaemia, can influence the expression and function of endothelin receptors after organ culture.

D-erythro-N,N-dimethylsphingosine inhibits bFGF-induced proliferation of cerebral, aortic and coronary smooth muscle cells.

The role of sphingosine kinase (SphK) on basic fibroblast growth factor (bFGF)-induced proliferation of cerebral, aortic and coronary smooth muscle cells (SMC) was addressed using D-erythro-N,N-dimethylsphingosine (DMS), an inhibitor of SphK which blocks conversion of sphingosine to sphingosine-1-phosphate (S1P). DMS concentration-dependently reduced the bFGF-induced proliferation of rat cerebral and aortic, and human coronary SMC. This suggests that SphK is one of the key enzymes in the mitogenic response to bFGF in vascular SMC as supported by the finding that S1P stimulated proliferation of SMC. Fumonisin B1, a dihydroceramidesynthase inhibitor which blocks the conversion of dihydrosphingosine to seramide, did not affect SMC proliferation induced by bFGF. Staurosporine, an inhibitor of protein kinase C (PKC), inhibited proliferation of SMC induced by bFGF, and both bFGF- and S1P-induced proliferation of SMC was sensitive to pertussis toxin (PTX), an inhibitor of Gi-protein activity. The present study thus demonstrates that SphK, PKC and Gi-protein activities are required for bFGF-mitogenic signaling in SMC. The bFGF mitogenic effect in vascular SMC might at least in part act via the SphK pathway and a Gi-protein.

Protein kinase C inhibition attenuates vascular ETB receptor upregulation and decreases brain damage after cerebral ischemia in rat

BackgroundProtein kinase C (PKC) is known to be involved in the pathophysiology of experimental cerebral ischemia. We have previously shown that after transient middle cerebral artery occlusion, there is an upregulation of endothelin receptors in the ipsilateral middle cerebral artery. The present study aimed to examine the effect of the PKC inhibitor Ro-32-0432 on endothelin receptor upregulation, infarct volume and neurology outcome after middle cerebral artery occlusion in rat.ResultsAt 24 hours after transient middle cerebral artery occlusion (MCAO), the contractile endothelin B receptor mediated response and the endothelin B receptor protein expression were upregulated in the ipsilateral but not the contralateral middle cerebral artery. In Ro-32-0432 treated rats, the upregulated endothelin receptor response was attenuated. Furthermore, Ro-32-0432 treatment decreased the ischemic brain damage significantly and improved neurological scores. Immunohistochemistry showed fainter staining of endothelin B receptor protein in the smooth muscle cells of the ipsilateral middle cerebral artery of Ro-32-0432 treated rats compared to control.ConclusionThe results suggest that treatment with Ro-32-0432 in ischemic stroke decreases the ischemic infarction area, neurological symptoms and associated endothelin B receptor upregulation. This provides a new perspective on possible mechanisms of actions of PKC inhibition in cerebral ischemia.

Increased monoaminergic neurotransmission improves compliance with physical activity recommendations in depressed patients with fatigue.

Clinical studies have shown that moderately intense physical activity effectively treats various types of depression. Beneficial effects have been reported in the acute phase of the disease as well as in a long-term perspective. In addition, epidemiological studies have shown that inactivity increases the risk of depression and that exercise prevents relapse. Depressed patients are often prescribed antidepressants, with or without psychotherapy. Some studies have, however, suggested that the most frequently used antidepressants, selective serotonin reuptake inhibitors (SSRIs), contribute to fatigue, which is a common residual symptom associated with depression and the target of the proposed study. Profound fatigue may in turn decrease the ability and motivation to perform the beneficial physical activity, e.g. via executive dysfunction. Fatigue and impaired executive function are commonly linked to disturbed cerebral dopaminergic and noradrenergic neurotransmission. This kind of dysfunction is hard to overcome, even when the major symptoms of depression are alleviated. Interestingly, physical activity has been suggested to improve the dopamine and norepinephrine neurotransmission. Furthermore, the favorable effects may be reciprocal; improved dopamine and norepinephrine transmission in the brain may hypothetically increase the ability and motivation to exercise, since some parts of the brain (e.g. the prefrontal cortex, striatum and cerebellum) that control movement and initiative receive dopaminergic and noradrenergic projections. Based on these findings and assumptions, our hypothesis is that increased dopaminergic and noradrenergic neurotransmission, via intake of a dopamine- and norepinephrine-enhancing agent, improves the compliance with prescribed physical activity in patients with depression and residual fatigue. We also believe that the increased physical activity can prevent relapse into depression, even after interruption of medication. Since increased physical activity also has been shown to improve executive cognitive function, we suggest that executive function should be examined as a secondary outcome together with other possibly related variables such as quality of life, sick leave and BMI.