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.
The endothelium of cerebral vessels produces a vasoactive signal substance endothelin, which has a strong and potent contractile effect. Endothelin has been suggested to play an important role in the pathogenesis of ischemic stroke.1–3⇓⇓ Two different subtypes of ET receptors mediate the vasomotor responses, the endothelin type A (ETA) and endothelin type B (ETB) receptors.4 The ETA receptor mediates a strong contractile effect in cerebral arteries, while the ETB receptor is mainly seen on the endothelial cells mediating vasodilatation via the release of nitric oxide.5–7⇓⇓ Recently we observed that there is a change in the endothelin receptor expression in ischemic stroke; there is a switch from the relaxant to the contractile phenotype of the ETB receptor. In addition, there is a selective upregulation of the ETB receptor after experimental focal cerebral ischemia as examined with both in vitro pharmacology and real-time polymerase chain reaction (PCR) in the rat middle cerebral artery (MCA).8 To examine this, we have developed a model to study endothelin receptor phenotype changes using organ culture. Incubation of cerebral arteries results in endothelin receptor changes that resemble the pattern seen in experimental ischemic stroke. Moreover, this upregulation can be further enhanced by cytokines.9,10⇓
However, the intracellular pathways responsible for the upregulation of ETB receptors in rat MCA in stroke as well as after organ culture need to be examined in further detail. It has been speculated that protein kinase C (PKC) may be involved in the control of transcription factors,11 and a study by Yonemochi et al12 revealed that PKC has effects on receptor modulation. Therefore, our aim was to examine the time course, the possible transcriptional and/or translational changes, and whether PKC is involved in the ETB receptor upregulation after organ culture in rat MCA.
The study was performed with the use of a sensitive in vitro myograph method in which endothelin-1 (ET-1) (ETA and ETB receptor agonist) and sarafotoxin 6c (S6c; selective ETB receptor agonist) were used to study the contractile responses mediated by the endothelin receptors.13 The relative endothelin receptor mRNA levels were measured with real-time PCR. The upregulation of ETB receptors in MCA after organ culture was attenuated by the transcriptional inhibitor actinomycin, suggesting that the change is due to de novo transcription of receptors. A possible role for PKC in this upregulation was examined with the use of the selective PKC inhibitor Ro-31-8220, which attenuated the induction of ETB receptor mRNA expression as well as the S6c-induced contraction.
Materials and Methods
Removal of Cerebral Vessels and Organ Culture Procedure
Male Wistar Hannover rats (Møllegaard Breeding Center, Copenhagen, Denmark), weighing 350 to 420 g, were used for the study. The animals were anesthetized with CO2 and decapitated. The brains were quickly removed and chilled in ice-cold bicarbonate buffer solution (for composition, see below). The right and left MCAs were removed.
The vessels used for in vitro pharmacology were either mounted in myographs immediately (fresh segments) or incubated for 6, 12, 24, or 48 hours at 37°C in humidified 5% CO2 and air in Dulbecco’s modified Eagle’s medium supplemented with penicillin (100 μg/mL) and thereafter mounted in myographs. When using inhibitors, they were added to the medium before 24 hours of incubation (actinomycin D, 80 μmol/L; cycloheximide, 36 μmol/L; Ro-31-8220, 1 μmol/L). For examination by real-time PCR, vessel segments were snap-frozen at −80°C.
In Vitro Pharmacology
A sensitive myograph was used for recording the isometric tension in isolated vessel segments.14,15⇓ The vessels, fresh or incubated, were cut into cylindrical segments, 0.5 mm long. The endothelium was removed mechanically by inserting a thin thread and rubbing the endothelium. The proper removal was checked by monitoring responses to 10−5 mol/L acetylcholine after ET-1 precontraction. The vessel segments were threaded on two 40-μm-diameter stainless steel wires and mounted in a Mulvany-Halpern myograph (Danish Myo Technology A/S). One wire was connected to a force displacement transducer attached to an analog-digital converter unit (ADInstruments). The other wire was attached to a movable displacement device allowing fine adjustments of vascular tension by varying the distance between the wires. The experiments were recorded on a computer by use of the software program Chart (ADInstruments). The segments were immersed in temperature-controlled (37°C) tissue baths containing a bicarbonate buffer solution of the following composition (mmol/L): NaCl 119, NaHCO3 15, KCl 4.6, MgCl2 1.2, NaH2PO4 1.2, CaCl2 1.5, and glucose 5.5. The solution was continuously gassed with 5% CO2 in O2, resulting in a pH of 7.4. The vessels were given an initial tension of 1.2 mN and were adjusted to this level of tension for 1 hour. The contractile capacity was determined by exposure to a potassium-rich (63.5 mmol/L) buffer solution with the same composition as the bicarbonate buffer solution except that NaCl was exchanged for KCl. The vessels were exposed to 63.5 mmol/L potassium twice, and the second contraction was used as a reference for the contractile capacity. Concentration-response curves for the agonists ET-1 and S6c were obtained by cumulative application of the substances (10−12 to 10−6.5 mol/L).
The vessels were snap-frozen at −80°C after removal, and total cellular RNA was extracted with the use of the FastRNA Kit Green (BIO 101) following the suppliers’ instructions. The resulting pellet was finally washed with ethanol, air-dried, and redissolved in 10 μL diethyl-pyrocarbonate–treated water. Reverse transcription of total RNA to cDNA was performed with the GeneAmp RNA PCR kit (PE Applied Biosystems) in a Perkin-Elmer DNA thermal cycler. First-strand cDNA was synthesized from 1 μg total RNA in a 20-μL reaction volume with random hexamers used as primers. The reaction mixture was incubated at 25°C for 10 minutes and 42°C for 15 minutes, heated to 99°C for 5 minutes, and chilled to 5°C for 5 minutes. Real-time PCR was performed in a GeneAmp 5700 Sequence Detection System (Perkin-Elmer, Applied Biosystems) with the GeneAmp SYBR Green kit (Perkin-Elmer, Applied Biosystems) with the cDNA synthesized above as template in a 50-μL reaction volume. A no-template control was included in all experiments. The GeneAmp 5700 Sequence Detection System monitors the growth of DNA in real time by an optics and imaging system via the binding of a fluorescent dye to double-stranded DNA. Specific primers for the rat ETA and ETB receptors were designed as follows: ETA receptor forward: 5′-ATTGCCCTCAGCGAACAC-3′; ETA receptor reverse: 5′-CAACCAAGCAGAAAGACGGTC-3′; ETB receptor forward: 5′-GATACGACAACTTCCGCTCCA-3′; ETB receptor reverse: 5′-GTCCACGATGAGGACAATGAG-3′.
Elongation factor-1 (EF-1) mRNA was used as a reference because it is the product of a housekeeping gene, continuously expressed to a constant amount in cells. The EF-1 primers were designed as follows: EF-1 forward: 5′-GCAAGCCCATGTGTG-TTGAA-3′; EF-1 reverse: 5′-TGATGACACCCACAGCAACTG-3′.
The real-time PCR was performed with the following profile: 50°C for 2 minutes and 95°C for 10 minutes, followed by 40 cycles with 95°C for 15 seconds and 60°C for 1 minute. To prove that the cDNA levels of EF-1 and the endothelin receptors were amplified with the same efficacy during real-time PCR, a standard curve was made in which the CT values were plotted against cDNA concentration on the basis of the following equation: CT=[lg (1+E)]−1lg(concentration), where E is the amplification efficiency with the optimal value of 1 (for further details, see Stenman et al8).
ET-1 and S6c (Auspep) were dissolved in 0.9% saline with 0.1% bovine serum albumin. Acetylcholine (Sigma) was dissolved in 0.9% saline. Ro-31-8220 (Calbiochem) was dissolved in dimethyl sulfoxide. Actinomycin D and cycloheximide (Sigma) were dissolved in redistilled water.
Polymerase Chain Reaction
Oligonucleotides and reagents for the PCR assay were purchased from Perkin-Elmer, Applied Biosystems.
Contractile experiments were performed on vessels that were incubated for 0, 6, 12, 24, and 48 hours and on vessels incubated for 24 hours with different inhibitors. The Emax values refer to maximum contraction, calculated as percentage of the contractile capacity of 63.5 mmol/L K+. Data are expressed as mean±SEM values, and n refers to number of vessel segments. There were 2 to 5 rats in each group, with 1 to 4 vessel segments from each. Statistical analyses were performed with 1-way ANOVA. P<0.05 was considered significant.
Polymerase Chain Reaction
PCR experiments were performed on fresh vessels and vessels incubated for 24 hours either with or without inhibitors; n refers to number of samples. Each sample contained 2 vessels to obtain a sufficient amount of tissue. The amount of ETA and ETB receptor mRNA was calculated as relative to the amount of EF-1 mRNA in the same sample by the following formula: X0/R0=2CtR−CtX, where X0 is original amount of endothelin receptor mRNA, R0 is original amount of EF-1 mRNA, CtR is CT value for EF-1, and CtX is CT value for the endothelin receptor. Statistical analyses were performed with paired Student’s t test; P<0.05 was considered significant.
In Vitro Pharmacology
K+-induced contractions did not differ significantly in the fresh vessel segments compared with the ones incubated for 6, 12, 24, and 48 hours, nor did they differ between vessel segments that were incubated 24 hours either with or without inhibitors (data not shown).
In fresh arteries, S6c did not induce contraction, but after organ culture there was a time-dependent increase in response (Figure 1, Table). There was no significant difference in Emax of the contractile response to ET-1 between fresh and cultured vessels. However, the EC50 value was significantly decreased after 48 hours of organ culture (Table).
In arteries cultured with the transcriptional inhibitor actinomycin D (80 μmol/L), the S6c-mediated contraction was significantly decreased after 24 hours compared with the 24-hour control to 10±13% of the K+-induced contraction, while the translational inhibitor cycloheximide (36 μmol/L) reduced the S6c-mediated contraction to an Emax of 57±4% (P<0.05; Figure 2).
Furthermore, the PKC inhibitor Ro-31-8220 (1 μmol/L) also attenuated contractile responses to S6c significantly after organ culture for 24 hours compared with the 24-hour control, with an Emax of 66±17% compared with the K+-induced contraction (P<0.05; Figure 3).
The 24-hour control was the same for all experiments, and the statistical analyses were performed with 1-way ANOVA.
The standard curves of each primer pair had almost similar slopes, indicating that the EF-1, ETA, and ETB cDNA levels were amplified with the same efficiency (data not shown). The values of each slope were close to 3.3, which means that the amplification efficiencies are almost optimal (E is very close to 1). In each PCR experiment a no-template control was included, and there were no signs of contaminating nucleic acids in those samples. The results from real-time PCR showed significantly elevated levels of ETB receptor mRNA relative to the amount of EF-1 mRNA in MCA after organ culture for 24 hours compared with fresh vessels, while the levels of ETA remained unchanged (ETA=0.022±0.007 and 0.022±0.003; P>0.05; ETB=0.055±0.011 and 0.009±0.001; P<0.05; Figure 4). Actinomycin D treatment resulted in significantly lower expression of ETB receptor mRNA in MCA after 24 hours of organ culture (ETB=0.009±0.004; P<0.05), as did Ro-31-8220 (ETB=0.020±0.007; P<0.05; Figure 5).
The study has shown a contractile effect of ET-1 (combined ETA and ETB receptor agonist) in fresh rat MCA, whereas S6c (specific ETB receptor agonist) had no contractile effect, suggesting that in fresh rat MCA the endothelin-induced contraction is mediated through ETA receptors exclusively. This is supported by findings showing that a specific ETA receptor antagonist blocks the ET-1 response in fresh vessels.10 After organ culture, the ETB receptors are upregulated, and their phenotype changes from a relaxant to a contractile type,9 as shown by the strong S6c-induced contraction. This is in accord with previous studies of rat mesenteric arteries13 and human omental arteries.16 We have recently demonstrated that this phenomenon of a local upregulation of contractile ETB receptors occurs in cerebral vessels after focal ischemia in the rat.8 The responses to the selective ETB agonist S6c was significantly stronger in the occluded MCA 48 hours after induction of stroke, while the responses to ET-1 in the same vessel were not altered. In addition, there was a selective increase in the ETB receptor mRNA expression after focal ischemia.8
The event of ischemic stroke may have an impact on intracellular pathways coupled to the transcription of both endothelin receptors in vascular smooth muscle cells in the affected hemisphere since we could in the present study show that the transcriptional blocker actinomycin D prevented this effect in vitro. Actinomycin D forms a complex with the DNA, blocking the RNA polymerase from binding and thereby preventing transcription.17 When arteries were cultured with actinomycin D, the contraction induced by S6c was almost completely abolished. This suggests that the S6c-induced contraction after 24 hours of organ culture was due to newly synthesized ETB receptors, and this is supported by the results from the real-time PCR in which the ETB receptor mRNA levels were significantly elevated after organ culture but attenuated after addition of actinomycin D. The translational inhibitor cycloheximide also attenuated the enhanced contractile response. This is in agreement with a study by Möller et al13 demonstrating that the upregulation of ETB receptors after organ culture of endothelium-denuded rat mesenteric arteries was mediated via increased transcription and subsequent translation of ETB receptor mRNA.
As in the human genome, the rat 5′-flanking region of the genes encoding the endothelin receptors contains several regulatory elements such as GATA motifs and E-boxes.18–21⇓⇓⇓ This indicates that the genes might be activated by, for example, inflammatory components after a focal ischemic stroke. An enhanced ETB receptor–mediated contraction of the rat basilar artery has been reported after incubation with the proinflammatory cytokines interleukin-1β and tumor necrosis factor-α,9 which thus supports the hypothesis that inflammatory components might be involved. One possible reason for the upregulation of endothelin receptors after stroke might be due to changes that occur in perfusion pressure during and after the occlusion. Cattaruzza et al22 presented data revealing that the ETB receptor mRNA levels in rat aortic smooth muscle cells were increased by up to 10-fold after periodic stretch. Furthermore, several studies have shown that there are increased levels of circulating ET-1 in ischemic stroke.1–3⇓⇓ These studies all suggest an important role of endothelin and endothelin receptors in the pathophysiological mechanisms in cerebral blood vessels after ischemic stroke.
The present study has focused further on the mechanisms involved in the changes seen in endothelin receptor expression in cerebral arteries. The PKC family, which comprises at least 12 cloned isozymes, plays a key role in many signaling cascades.23 Wang and Dhalla11 have suggested a possible role for PKC in the modulation of transcription factors. There is also evidence that blocking PKC can inhibit β-adrenoceptor upregulation after treatment with angiotensin-converting enzyme inhibitors.12 In our study we show that organ culture with the PKC inhibitor Ro-31-8220 attenuates the S6c-induced contraction in rat MCA, pointing out the critical role for PKC in the upregulation of ETB receptors. Ro-31-8220 is an inhibitor of PKC-α, -β, -γ, and -ε isoforms, but it has also been shown to block the effects of mitogen-activated protein kinase (MAPK) activated protein (MAPKAP) K1β and p70 S624 as well as the expression of MAPK P-1.25 Thus, the upregulation of ETB receptors in rat MCA after organ culture may not necessarily be PKC dependent only. Our next target will be to further elucidate the role of PKC in the receptor upregulation and to examine whether a specific isoform of the enzyme is responsible. This new knowledge will be applied in our model of experimental ischemic stroke to further investigate the upregulation of ETB receptors and their possible role in the pathophysiology of ischemic stroke.
This study was supported by a grant from the Swedish Research Council (grant No. 5958) and the Swedish Heart and Lung Foundation.
- Received July 15, 2002.
- Revision received January 7, 2003.
- Accepted January 13, 2003.
- ↵Lampl Y, Fleminger G, Gilad R, Galron R, Sarova-Pinhas I, Sokolovsky M. Endothelin in cerebrospinal fluid and plasma of patients in the early stage of ischemic stroke. Stroke. 1997; 28: 1951–1955.
- ↵Ziv I, Fleminger G, Djaldetti R, Achiron A, Melamed E, Sokolovsky M. Increased plasma endothelin-1 in acute ischemic stroke. Stroke. 1992; 23: 1014–1016.
- ↵de Nucci G, Thomas R, D’Orleans-Juste P, Antunes E, Walder C, Warner TD, Vane JR. Pressor effects of circulating endothelin are limited by its removal in the pulmonary circulation and by the release of prostacyclin and endothelium-derived relaxing factor. Proc Natl Acad Sci U S A. 1988; 85: 9797–9800.
- ↵Stenman E, Malmsjö M, Uddman E, Gidö G, Wieloch T, Edvinsson L. Cerebral ischemia upregulates vascular endothelin ETB receptors in rat. Stroke. 2002; 33: 2311–2316.
- ↵Yonemochi H, Yasunaga S, Teshima Y, Iwao T, Akiyoshi K, Nakagawa M, Saikawa T, Ito M. Mechanism of beta-adrenergic receptor upregulation induced by ACE inhibition in cultured neonatal rat cardiac myocytes: roles of bradykinin and protein kinase C. Circulation. 1998; 97: 2268–2273.
- ↵Mulvany MJ, Halpern W. Contractile properties of small arterial resistance vessels in spontaneously hypertensive and normotensive rats. Circ Res. 1977; 41: 19–26.
- ↵Arai H, Nakao K, Takaya K, Hosoda K, Ogawa Y, Nakanishi S, Imura H. The human endothelin-B receptor gene: structural organization and chromosomal assignment. J Biol Chem. 1993; 268: 3463–3470.
- ↵Hosoda K, Nakao K, Tamura N, Arai H, Ogawa Y, Suga S, Nakanishi S, Imura H. Organization, structure, chromosomal assignment, and expression of the gene encoding the human endothelin-A receptor. J Biol Chem. 1992; 267: 18797–18804.
- ↵Prody C. Direct submission to GenBank. C. Prody, Hospital for Sick Children, Cardiovascular Research, 555 University Ave, Toronto, Ontario M5G 1X8, Canada. 1995.
- ↵Cattaruzza M, Dimigen C, Ehrenreich H, Hecker M. Stretch-induced endothelin B receptor-mediated apoptosis in vascular smooth muscle cells. FASEB J. 2000; 14: 991–998.
- ↵Beltman J, McCormick F, Cook SJ. The selective protein kinase C inhibitor, Ro-31-8220, inhibits mitogen-activated protein kinase phosphatase-1 (MKP-1) expression, induces c-Jun expression, and activates Jun N-terminal kinase. J Biol Chem. 1996; 271: 27018–27024.