Hilda Ahnstedt

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.

Human cerebrovascular contractile receptors are upregulated via a B-Raf/MEK/ERK-sensitive signaling pathway

BackgroundCerebral ischemia results in a rapid increase in contractile cerebrovascular receptors, such as the 5-hydroxytryptamine type 1B (5-HT1B), angiotensin II type 1 (AT1), and endothelin type B (ETB) receptors, in the vessel walls within the ischemic region, which further impairs local blood flow and aggravates tissue damage. This receptor upregulation occurs via activation of the mitogen-activated protein kinase pathway. We therefore hypothesized an important role for B-Raf, the first signaling molecule in the pathway. To test our hypothesis, human cerebral arteries were incubated at 37°C for 48 h in the absence or presence of a B-Raf inhibitor: SB-386023 or SB-590885. Contractile properties were evaluated in a myograph and protein expression of the individual receptors and activated phosphorylated B-Raf (p-B-Raf) was evaluated immunohistochemically.Results5-HT1B, AT1, and ETB receptor-mediated contractions were significantly reduced by application of SB-590885, and to a smaller extent by SB-386023. A marked reduction in AT1 receptor immunoreactivity was observed after treatment with SB-590885. Treatment with SB-590885 and SB-386023 diminished the culture-induced increase of p-B-Raf immunoreactivity.ConclusionsB-Raf signaling has a key function in the altered expression of vascular contractile receptors observed after organ culture. Therefore, specific targeting of B-Raf might be a novel approach to reduce tissue damage after cerebral ischemia by preventing the previously observed upregulation of contractile receptors in smooth muscle cells.

U0126 attenuates cerebral vasoconstriction and improves long-term neurologic outcome after stroke in female rats.

Sex differences are well known in cerebral ischemia and may impact the effect of stroke treatments. In male rats, the MEK1/2 inhibitor U0126 reduces ischemia-induced endothelin type B (ETB) receptor upregulation, infarct size and improves acute neurologic function after experimental stroke. However, responses to this treatment in females and long-term effects on outcome are not known. Initial experiments used in vitro organ culture of cerebral arteries, confirming ERK1/2 activation and increased ETB receptor-mediated vasoconstriction in female cerebral arteries. Transient middle cerebral artery occlusion (tMCAO, 120 minutes) was induced in female Wistar rats, with U0126 (30 mg/kg intraperitoneally) or vehicle administered at 0 and 24 hours of reperfusion, or with no treatment. Infarct volumes were determined and neurologic function was assessed by 6-point and 28-point neuroscores. ETB receptor-mediated contraction was studied with myograph and protein expression with immunohistochemistry. In vitro organ culture and tMCAO resulted in vascular ETB receptor upregulation and activation of ERK1/2 that was prevented by U0126. Although no effect on infarct size, U0126 improved the long-term neurologic function after experimental stroke in female rats. In conclusion, early prevention of the ERK1/2 activation and ETB receptor-mediated vasoconstriction in the cerebral vasculature after ischemic stroke in female rats improves the long-term neurologic outcome.

Differential localization and characterization of functional calcitonin gene-related peptide receptors in human subcutaneous arteries.

Calcitonin gene‐related peptide (CGRP) and its receptor are widely distributed within the circulation and the mechanism behind its vasodilation not only differs from one animal species to another but is also dependent on the type and size of vessel. The present study examines the nature of CGRP‐induced vasodilation, characteristics of the CGRP receptor antagonist telcagepant and localization of the key components calcitonin receptor‐like receptor (CLR) and receptor activity modifying protein 1 (RAMP1) of the CGRP receptor in human subcutaneous arteries.

Involvement of calcium-calmodulin-dependent protein kinase II in endothelin receptor expression in rat cerebral arteries

Experimental cerebral ischemia and organ culture of cerebral arteries result in the enhanced expression of endothelin ET(B) receptors in smooth muscle cells via increased transcription. The present study was designed to evaluate the involvement of calcium-calmodulin-dependent protein kinase (CAMK) in the transcriptional expression of endothelin receptors after organ culture. Rat basilar arteries were incubated for 24 h with or without the CAMK inhibitor KN93 or ERK1/2 inhibitor U0126. The contractile responses to endothelin-1 (ET-1; ET(A) and ET(B) receptor agonist) and sarafotoxin 6c (S6c; ET(B) receptor agonist) were studied using a sensitive myograph. The mRNA levels of the ET(A) and ET(B) receptors and CAMKII were determined by real-time PCR, and their protein levels were evaluated by immunohistochemistry and Western blot. The mRNA levels of CAMKII and the ET(B) receptor increased during organ culture, but there was no change in the expression of the ET(A) receptor. This effect was abolished by coincubation with KN93 or U0126. In functional studies, both inhibitors attenuated the S6c-induced contraction. Incubating the arteries with KN93, but not U0126, decreased the amount of phosphorylated CAMKII. The inhibitors had no effect on the levels of myosin light chain during organ culture, as measured by Western blot. CAMKII is involved in the upregulation of the endothelin ET(B) receptor and interacts with the ERK1/2 pathway to enhance receptor expression. CAMKII has no effect on the contractile apparatus in rat cerebral arteries.

Male-Female Differences in Upregulation of Vasoconstrictor Responses in Human Cerebral Arteries

Background and purpose Male-female differences may significantly impact stroke prevention and treatment in men and women, however underlying mechanisms for sexual dimorphism in stroke are not understood. We previously found in males that cerebral ischemia upregulates contractile receptors in cerebral arteries, which is associated with lower blood flow. The present study investigates if cerebral arteries from men and women differ in cerebrovascular receptor upregulation. Experimental approach Freshly obtained human cerebral arteries were placed in organ culture, an established model for studying receptor upregulation. 5-hydroxtryptamine type 1B (5-HT1B), angiotensin II type 1 (AT1) and endothelin-1 type A and B (ETA and ETB) receptors were evaluated using wire myograph for contractile responses, real-time PCR for mRNA and immunohistochemistry for receptor expression. Key results Vascular sensitivity to angiotensin II and endothelin-1 was markedly lower in cultured cerebral arteries from women as compared to men. ETB receptor-mediated contraction occurred in male but not female arteries. Interestingly, there were similar upregulation in mRNA and expression of 5-HT1B, AT1, and ETB receptors and in local expression of Ang II after organ culture. Conclusions and Implications In spite of receptor upregulation after organ culture in both sexes, cerebral arteries from women were significantly less responsive to vasoconstrictors angiotensin II and endothelin-1 as compared to arteries from men. This suggests receptor coupling and/or signal transduction mechanisms involved in cerebrovascular contractility may be suppressed in females. This is the first study to demonstrate sex differences in the vascular function of human brain arteries.

Characterization of Relaxant Responses to Natriuretic Peptides in the Human Microcirculation In Vitro and In Vivo

We characterized the vasodilatory effects of ANP, BNP, and CNP in human subcutaneous arterioles in vitro and the cutaneous microcirculation in vivo.

Inositol 1,4,5-Trisphosphate Receptor and Calcium Calmodulin-Dependent Protein Kinase are Involved in Endothelin Receptor Expression in Rat Cerebral Artery

Background: The present study was designed to examine the influence of inositol 1,4,5-trisphosphate receptor (IP3R) and calcium calmodulin-dependent protein kinase (CaMK) on endothelin receptor regulation during organ culture in rat middle cerebral arteries (MCA). Methods: MCA segments were incubated with or without xestospongin C (XeC), an IP3R inhibitor, or KN93, a CaMKII blocker. The mRNA levels of the ETA and ETB receptors, nuclear factor of activated T cells activating protein (NFam1), CaMKII, IP3R and Downstream regulatory element antagonist modulator (DREAM) protein were determined by real-time PCR or immunohistochemistry. Contractile responses to endothelin-1 (ET-1) and sarafotoxin 6c (S6c) were studied by a sensitive myograph and the intracellular calcium levels [Ca2+]i were evaluated by the FURA-2AM. Results: The contractile responses to ET-1 and S6c, the ETB receptors mRNA level and the baseline level of intracellular calcium [Ca2+]i were all increased after 24 h organ culture. Incubation with XeC or KN93 attenuated ETB receptor expression and increase in [Ca2+]i induced by ET-1 or S6c. XeC decreased the NFam1 mRNA levels while KN93 decreased DREAM protein levels. Conclusions: The study suggests that CaMKII and calcium release via IP3R are involved in the mechanisms regulate ETB receptor putatively via DREAM and NFam1, respectively.

Intracellular calcium levels and vasoconstriction induced by 5-CT and s6c in rat after cerebral ischemia

Objectives: Statin reduces cerebrovascular events independent of its cholesterol lowering effect. We hypothesized statin inhibits early atherosclerotic change in common carotid artery (CCA), and investigated its effect on lectin-like oxidized-LDL receptor-1 (LOX-1) and monocyte chemoattractant protein-1 (MCP-1) expression, both of which are early atherosclerotic markers. Methods: Stroke-prone spontaneous hypertensive rats (SHR-SP) were divided into 3 groups. Each group was treated with vehicle or simvastatin for 2 or 4 weeks and fed high fatty food and 1% NaCl water for 2 weeks, and CCA was removed. LOX-1 and MCP-1 expression as well as macrophage infiltration were histologically investigated. Lipid deposition was also investigated by Sudan III staining. Results: Simvastatin groups showed significantly smaller amount of lipid deposition and LOX-1 and MCP-1 expressions, independent of serum lipid levels. Macrophage infiltration was also inhibited. Conclusions: Simvastatin suppresed atherosclerotic change in CCA of hypertensive rats with high fatty diet. Reduction of cerebrovascular event by statins may be brought by the direct inhibition against atherosclerotic change.

Impact of cytokines and growth factors on contractile endothelin receptors in rat cerebral arteries

Introduction: One approach of increasing mean arterial pressure (MAP), in order to secure a pressure gradient for vital organs, is provided by the use of vasopressor agents. The consequence of augmenting MAP by elevating vascular resistance or cardiac output (CO) on blood flow and oxygenation of the brain is not clear. More specifically, the influence of noradrenaline (NA), which is an a-agonist causing vasoconstriction, on cerebral hemodynamics is ambiguous. Furthermore, the influence of NA on cerebral oxygenation has not been investigated. Aim: The aim of this study was to evaluate the impact of the infusion of NA on cerebral oxygenation in healthy subjects. Methods: Three doses of NA (0.05, 0.1 and 0.15 mgkg 1 mins 1 for 20mins each) were infused in nine healthy subjects (6 males; 26±6 years old, mean±s.d.). MAP, cerebral oxygenation characterized by frontal lobe oxygenation (ScO2) and jugular venous oxygen saturation (SjvO2), middle cerebral artery mean flow velocity (MCA Vmean), cardiac output (CO) and arterial pressure for carbon dioxide (PaCO2) were evaluated. Results: MAP increased from 88 (22) [median (range)] to 115 (30) mm Hg with increasing doses of NA (P < 0.05 for 0.1 and 0.15 mg kg 1 mins 1 of NA) and reflected an increase in total peripheral resistance (20.3 (13.7) to 25.2 (12.1) mm Hg 1/L mins ; P < 0.05) as CO remained at a baseline value (Table). ScO2 and SjvO2 lowered with increasing doses of NA, reaching statistical significance with NA infused at 0.1 mg kg 1 mins 1 and higher (ScO2: 78 (19)% to 69 (22)%; P < 0.05; SjvO2: 67%±8% to 64%±7%; P < 0.01). MCA Vmean was reduced with each doses of NA (56.9±11.2 to 55.0±11.7 cm secs ; P < 0.05). Finally, PaCO2 lowered with increasing doses of NA (5.4±0.4 to 5.1±0.4 kPa; P < 0.001). Conclusion: This study suggests that infusion of 0.1 mg kg 1 mins 1 and higher doses of NA negatively affects cerebral oxygenation as it increases MAP by peripheral vasoconstriction.