Characterization of Adrenoceptors in the Vessel Wall and in Cultured Vascular Wall Cells of the Porcine Aorta
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1993
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| author | Erdbrügger, Wilhelm |
| author_facet | Erdbrügger, Wilhelm |
| contents | <p>Using the porcine aorta as an example, the expression of adrenoceptors in cultured SMCs and in vascular muscle was investigated with the help of radioligand binding studies. [<sup>3</sup>H]-prazosin for α1-, [<sup>3</sup>H]-yohimbine for α2-, and [<sup>125</sup>J]-cyanopindolol for ß-adrenoceptors was used.</p> <p>It has been shown that adrenoceptors in the vascular muscle (media) of porcine aorta is composed of about 23% of α1- (165 fmol/mg protein), 75% of α2- (548 fmol/mg protein) and only 2% ß-adrenoceptors (15 fmol/mg protein).</p> <p>In further investigations, the occurrence of four different adrenoreceptor subtypes in the vascular muscle could be detected. The α1-adrenoceptors apparently consist of a homogeneous population of α1B-adrenoceptors. On the other hand, about 70% of α2A-adrenoceptors and 30% α2B-like adrenoceptors were found and addition, only 2% ß2-adrenoceptors are present. However, the ß2-selective antagonist ICI 188.551 has an unusually low affinity for ß2-adrenoceptors in the media of porcine aortas (Kl 130 nM). ICI 188.551 also appears to have a similarly low affinity for ß2-adrenoceptors of cultured ECs from pig aortas (Kl 110 nM).</p> <p>[<sup>125</sup>J]-cyanopindolol bound in vascular muscle membranes of the media except at ß2- adrenoceptors (KD 10 pM) to a second low-affinity binding site (KD > 100 nM). However, the assumption that "atypical" ß-adrenoceptors may be present in porcine aorta SMCs as well as in intestinal SMCs was not confirmed. Thus, the second binding site was replaced by (+) isoprenaline, (-) adrenaline and (-) noradrenaline with the same affinity and displaced only in millimolar concentrations. In addition, no stereoselectivity was detected for (-) -isoprenaline.</p> <p>In radioligand binding studies on membrane preparations from cultured porcine aorta SMCs, a specific radioligand binding was detected for [3H]-prazosin, [3H]-yohimbine and [<sup>125</sup>J]-cyanopindolol. For all three radioligands examined, however, the non-specific binding accounted for more than 85% of the total bond. For methodological reasons, therefore, a quantitative analysis of adrenoceptors on cultured SMCs was not possible by radioligand binding studies. Therefore, with the help of the intracellular Ca2+ indicator Fura-2, it was investigated whether adrenoceptors, as already described for NPY receptors, cause changes in the Cai in cultured SMCs.</p> <p>Intracellular Ca<sup>2+</sup> increases in cultured SMCs were stimulated with noradrenaline or the co-transmitter NPY, this led to a temporary increase in intracellular calcium concen­tration (Cai).</p> <p>Using selective agonists and antagonists, it could be shown that the Cai increases in the cells caused by noradrenaline are mainly caused by α2-adrenoceptors (presumably α2A- and α2B-like adrenoceptors) and only to a small extent by αl-adrenoceptors (presumably α1B-adrenoceptors).</p> <p>The Cai increases mediated by α1 - and α2-adrenoceptors were completely inhibited by the pretreatment of cultured SMCs with pertussis toxin. Cai increases due to angiotensin II, on the other hand, were not inhibited. Presumably, therefore, both α-adrenoceptors cause Cai increases in the cells by coupling to a pertussis toxin sensitive G-protein. However, while the stimulation of α2-adrenoceptors led exclusively to a mobilization of Ca2+ from intracellular stores, about 50% of the Cai response stimulated by the α1-adrenoreceptor agonist phenylephrine was due to an influx of extracellular Ca<sup>2+</sup>.</p> <p>The increases in Cai stimulated by NPY were also pertussis toxin sensitive. Studies with NPY-analogue agonists showed that the Cai increases were probably mediated by Y3-like NPY receptors. For example, peptide YY (PYY) elicited only about 55% of the Ca<sup>2+</sup> response stimulated by NPY at the same concentration. NPY-mediated Ca<sup>2+</sup> increases decreased by about half in the absence of extracellular Ca2+ and appeared to be caused in roughly equal parts by intracellular Ca<sup>2+</sup> mobilization and extracellular Ca<sup>2+</sup> influx. However, organic Ca<sup>2+</sup> channel antagonists such as dihydropyridines like verapamil, diltiazem or nifedipine had no influence on the Ca<sup>2+</sup> response induced by NPY.</p> <p>The vascular contractions in arteries induced by α2-adrenoceptors and NPY receptors are mediated by a pertussis toxin-sensitive G-proteins in cultured SMCs, like the Ca2+ increases. However, for vascular contraction by α2-adrenoceptors and NPY receptors, an influx of extracellular Ca<sup>2+</sup> is essential, which can be inhibited by dihydropyridines. However, such Ca<sup>2+</sup> influx was not observed in cultured SMCs after stimulation with the α2-selective agonist α-methyl-noradrenalin or NPY. Dihydropyridine-sensitive Ca<sup>2+</sup> channels in vascular muscle cells are, for example, voltage-gated. In cultured SMCs from porcine aortas, voltage-gated Ca<sup>2+</sup> channels appear to be either completely absent or decoupled. The depolarization of the cells with 50 mM KCI did not lead to any measurable changes in the intracellular Ca<sup>2+</sup> concentration.</p> <p>Arachidonic acid metabolism of cultured vascular wall cells was investigated since prostaglandins, metabolites of arachidonic acid, are locally active vasoactive factors that can be formed after hormonal or mechanical stimulation of ECs but also of SMCs. ­Studies on cultured endothelial and SMCs from porcine aortas showed that radioactive arachidonic acid was predominantly incorporated into the membrane phospholipids phosphatidylcholine, phosphatidyl­inositol, phosphatidylserine and phosphatid­yl­etha­nol­­amine. Only a small proportion was metabolized into prostaglandins. Prostacyclin (PGI<sub>2</sub>), PGF<sub>2α</sub> or PGE<sub>2</sub> were synthesized by both cell types only after stimulation.</p> <p>After mechanical stimulation or stimulation with the Ca2+ ionophore A23187, SMCs synthesized predominantly PGE<sub>2</sub> (about 80%). In contrast, in stimulation supernatants of ECs, PGF<sub>2α</sub> and 6-oxo- PGF<sub>1α</sub> the stable degradation product of the vasodilator PGI<sub>2</sub> could be detected in addition to comparable amounts of PGE<sub>2</sub>. ECs stimulated with the Ca2+ ionophore A23187 predominantly released the vasoconstrictor PGF<sub>2α</sub> (about 60%) and only about half as much PGI<sub>2</sub>. Mechanically stimulated ECs, on the other hand, produced predominantly PGI<sub>2</sub> (56%) and significantly less PGF<sub>2α</sub> (29%). This behavior of cultured ECs could explain different, stimulus-dependent endothelial effects on vascular tone. Thus, an increase in shear forces in various arteries leads to endothelium-dependent vascular relaxation. The Ca<sup>2+</sup> ionophore A23187, on the other hand, can stimulate the formation of vasoconstricting cyclooxygenase products in some tissues. Whether these findings are related to the behavior observed in cultured ECs to synthesize predominantly the vasodilator PGI<sub>2</sub> on mechanical stimulation and predominantly the vasoconstrictor PGF<sub>2α</sub> after stimulation with A23187 still needs to be clarified.</p> <p>In cultured SMCs, the effect of prostaglandins on α2-adrenoreceptor-mediated Ca<sup>2+</sup> increases was investigated. The prostaglandin metabolism of SMCs showed no interaction with the Cai caused by α-methyl-noradrenaline. Neither the pretreatment of SMCs with the cyclooxygenase inhibitor indomethacin, nor with the lipoxygenase inhibitor nordihydroguaretic acid (NDGA) or arachidonic acid changed the level or course of the Cai response stimulated by α2-adrenoceptors. Iloprost, a stable analogue of prostacyclin (PGI<sub>2</sub>), did not affect the (α 2-adrenoreceptor mediated Cai increases in SMCs. Only high concentrations of PGE<sub>2</sub> (10 µM) led to a partial inhibition (30%) of the Ca<sup>2+</sup> increases stimulated by α-methyl-noradrenaline. The stimulation of adenylate cyclase by forskolin or the pre-induction of cells with stable derivatives of the "second messenger" cAMP and cGMP, which are associated with the relaxation of SMCs, did not lead to any change in the Ca<sup>2+</sup> response. NPY-stimulated Ca<sup>2+</sup> increases were also not affected by pre-incubation with PP56, an inhibitor of NPY-stimulated vasoconstriction in arteries.</p> <p>If the transient Ca<sup>2+</sup> increases by α2-adreno- and NPY receptors observed in cultured SMCs are also important in vascular muscle, it is likely that vascular relaxation by PGI<sub>2</sub> or PGE<sub>2</sub> or the action of PP56 occurs via mechanisms that are downstream of or independent of the transient Ca<sup>2+</sup> response.</p> <p>PGF<sub>2α</sub> itself stimulated Ca<sup>2+</sup> increases in cultured SMCs. All four investigated vasoconstrictors noradrenaline, NPY, angiotensin II and PGF<sub>2α</sub> stimulated a transient Ca<sup>2+</sup> response with a similar time course in the cultured muscle cells. Therefore, receptors that otherwise mediate vasoconstriction in vascular muscle cells are apparently also expressed in the cultured, phenotypically modulated SMCs. However, cultured SMCs and cells in vascular muscle seem to differ in the mechanism of the Ca2+ response by α2- adrenoceptors and NPY receptors. It is possible that these changes are typical of proliferating SMCs, which are increasingly found in arteriosclerotic vessels under pathophysiological conditions.</p> |
| format | Recurso digital |
| id | zenodo_https___doi_org_10_5281_zenodo_14697832 |
| institution | Zenodo |
| language | eng |
| publishDate | 1993 |
| publisher | Zenodo |
| record_format | zenodo |
| spellingShingle | Characterization of Adrenoceptors in the Vessel Wall and in Cultured Vascular Wall Cells of the Porcine Aorta Erdbrügger, Wilhelm Porcine Aorta Adrenoreceptors Endothelial Cells Smooth Muscle Cells Valcular Tone Prostaglandins <p>Using the porcine aorta as an example, the expression of adrenoceptors in cultured SMCs and in vascular muscle was investigated with the help of radioligand binding studies. [<sup>3</sup>H]-prazosin for α1-, [<sup>3</sup>H]-yohimbine for α2-, and [<sup>125</sup>J]-cyanopindolol for ß-adrenoceptors was used.</p> <p>It has been shown that adrenoceptors in the vascular muscle (media) of porcine aorta is composed of about 23% of α1- (165 fmol/mg protein), 75% of α2- (548 fmol/mg protein) and only 2% ß-adrenoceptors (15 fmol/mg protein).</p> <p>In further investigations, the occurrence of four different adrenoreceptor subtypes in the vascular muscle could be detected. The α1-adrenoceptors apparently consist of a homogeneous population of α1B-adrenoceptors. On the other hand, about 70% of α2A-adrenoceptors and 30% α2B-like adrenoceptors were found and addition, only 2% ß2-adrenoceptors are present. However, the ß2-selective antagonist ICI 188.551 has an unusually low affinity for ß2-adrenoceptors in the media of porcine aortas (Kl 130 nM). ICI 188.551 also appears to have a similarly low affinity for ß2-adrenoceptors of cultured ECs from pig aortas (Kl 110 nM).</p> <p>[<sup>125</sup>J]-cyanopindolol bound in vascular muscle membranes of the media except at ß2- adrenoceptors (KD 10 pM) to a second low-affinity binding site (KD > 100 nM). However, the assumption that "atypical" ß-adrenoceptors may be present in porcine aorta SMCs as well as in intestinal SMCs was not confirmed. Thus, the second binding site was replaced by (+) isoprenaline, (-) adrenaline and (-) noradrenaline with the same affinity and displaced only in millimolar concentrations. In addition, no stereoselectivity was detected for (-) -isoprenaline.</p> <p>In radioligand binding studies on membrane preparations from cultured porcine aorta SMCs, a specific radioligand binding was detected for [3H]-prazosin, [3H]-yohimbine and [<sup>125</sup>J]-cyanopindolol. For all three radioligands examined, however, the non-specific binding accounted for more than 85% of the total bond. For methodological reasons, therefore, a quantitative analysis of adrenoceptors on cultured SMCs was not possible by radioligand binding studies. Therefore, with the help of the intracellular Ca2+ indicator Fura-2, it was investigated whether adrenoceptors, as already described for NPY receptors, cause changes in the Cai in cultured SMCs.</p> <p>Intracellular Ca<sup>2+</sup> increases in cultured SMCs were stimulated with noradrenaline or the co-transmitter NPY, this led to a temporary increase in intracellular calcium concen­tration (Cai).</p> <p>Using selective agonists and antagonists, it could be shown that the Cai increases in the cells caused by noradrenaline are mainly caused by α2-adrenoceptors (presumably α2A- and α2B-like adrenoceptors) and only to a small extent by αl-adrenoceptors (presumably α1B-adrenoceptors).</p> <p>The Cai increases mediated by α1 - and α2-adrenoceptors were completely inhibited by the pretreatment of cultured SMCs with pertussis toxin. Cai increases due to angiotensin II, on the other hand, were not inhibited. Presumably, therefore, both α-adrenoceptors cause Cai increases in the cells by coupling to a pertussis toxin sensitive G-protein. However, while the stimulation of α2-adrenoceptors led exclusively to a mobilization of Ca2+ from intracellular stores, about 50% of the Cai response stimulated by the α1-adrenoreceptor agonist phenylephrine was due to an influx of extracellular Ca<sup>2+</sup>.</p> <p>The increases in Cai stimulated by NPY were also pertussis toxin sensitive. Studies with NPY-analogue agonists showed that the Cai increases were probably mediated by Y3-like NPY receptors. For example, peptide YY (PYY) elicited only about 55% of the Ca<sup>2+</sup> response stimulated by NPY at the same concentration. NPY-mediated Ca<sup>2+</sup> increases decreased by about half in the absence of extracellular Ca2+ and appeared to be caused in roughly equal parts by intracellular Ca<sup>2+</sup> mobilization and extracellular Ca<sup>2+</sup> influx. However, organic Ca<sup>2+</sup> channel antagonists such as dihydropyridines like verapamil, diltiazem or nifedipine had no influence on the Ca<sup>2+</sup> response induced by NPY.</p> <p>The vascular contractions in arteries induced by α2-adrenoceptors and NPY receptors are mediated by a pertussis toxin-sensitive G-proteins in cultured SMCs, like the Ca2+ increases. However, for vascular contraction by α2-adrenoceptors and NPY receptors, an influx of extracellular Ca<sup>2+</sup> is essential, which can be inhibited by dihydropyridines. However, such Ca<sup>2+</sup> influx was not observed in cultured SMCs after stimulation with the α2-selective agonist α-methyl-noradrenalin or NPY. Dihydropyridine-sensitive Ca<sup>2+</sup> channels in vascular muscle cells are, for example, voltage-gated. In cultured SMCs from porcine aortas, voltage-gated Ca<sup>2+</sup> channels appear to be either completely absent or decoupled. The depolarization of the cells with 50 mM KCI did not lead to any measurable changes in the intracellular Ca<sup>2+</sup> concentration.</p> <p>Arachidonic acid metabolism of cultured vascular wall cells was investigated since prostaglandins, metabolites of arachidonic acid, are locally active vasoactive factors that can be formed after hormonal or mechanical stimulation of ECs but also of SMCs. ­Studies on cultured endothelial and SMCs from porcine aortas showed that radioactive arachidonic acid was predominantly incorporated into the membrane phospholipids phosphatidylcholine, phosphatidyl­inositol, phosphatidylserine and phosphatid­yl­etha­nol­­amine. Only a small proportion was metabolized into prostaglandins. Prostacyclin (PGI<sub>2</sub>), PGF<sub>2α</sub> or PGE<sub>2</sub> were synthesized by both cell types only after stimulation.</p> <p>After mechanical stimulation or stimulation with the Ca2+ ionophore A23187, SMCs synthesized predominantly PGE<sub>2</sub> (about 80%). In contrast, in stimulation supernatants of ECs, PGF<sub>2α</sub> and 6-oxo- PGF<sub>1α</sub> the stable degradation product of the vasodilator PGI<sub>2</sub> could be detected in addition to comparable amounts of PGE<sub>2</sub>. ECs stimulated with the Ca2+ ionophore A23187 predominantly released the vasoconstrictor PGF<sub>2α</sub> (about 60%) and only about half as much PGI<sub>2</sub>. Mechanically stimulated ECs, on the other hand, produced predominantly PGI<sub>2</sub> (56%) and significantly less PGF<sub>2α</sub> (29%). This behavior of cultured ECs could explain different, stimulus-dependent endothelial effects on vascular tone. Thus, an increase in shear forces in various arteries leads to endothelium-dependent vascular relaxation. The Ca<sup>2+</sup> ionophore A23187, on the other hand, can stimulate the formation of vasoconstricting cyclooxygenase products in some tissues. Whether these findings are related to the behavior observed in cultured ECs to synthesize predominantly the vasodilator PGI<sub>2</sub> on mechanical stimulation and predominantly the vasoconstrictor PGF<sub>2α</sub> after stimulation with A23187 still needs to be clarified.</p> <p>In cultured SMCs, the effect of prostaglandins on α2-adrenoreceptor-mediated Ca<sup>2+</sup> increases was investigated. The prostaglandin metabolism of SMCs showed no interaction with the Cai caused by α-methyl-noradrenaline. Neither the pretreatment of SMCs with the cyclooxygenase inhibitor indomethacin, nor with the lipoxygenase inhibitor nordihydroguaretic acid (NDGA) or arachidonic acid changed the level or course of the Cai response stimulated by α2-adrenoceptors. Iloprost, a stable analogue of prostacyclin (PGI<sub>2</sub>), did not affect the (α 2-adrenoreceptor mediated Cai increases in SMCs. Only high concentrations of PGE<sub>2</sub> (10 µM) led to a partial inhibition (30%) of the Ca<sup>2+</sup> increases stimulated by α-methyl-noradrenaline. The stimulation of adenylate cyclase by forskolin or the pre-induction of cells with stable derivatives of the "second messenger" cAMP and cGMP, which are associated with the relaxation of SMCs, did not lead to any change in the Ca<sup>2+</sup> response. NPY-stimulated Ca<sup>2+</sup> increases were also not affected by pre-incubation with PP56, an inhibitor of NPY-stimulated vasoconstriction in arteries.</p> <p>If the transient Ca<sup>2+</sup> increases by α2-adreno- and NPY receptors observed in cultured SMCs are also important in vascular muscle, it is likely that vascular relaxation by PGI<sub>2</sub> or PGE<sub>2</sub> or the action of PP56 occurs via mechanisms that are downstream of or independent of the transient Ca<sup>2+</sup> response.</p> <p>PGF<sub>2α</sub> itself stimulated Ca<sup>2+</sup> increases in cultured SMCs. All four investigated vasoconstrictors noradrenaline, NPY, angiotensin II and PGF<sub>2α</sub> stimulated a transient Ca<sup>2+</sup> response with a similar time course in the cultured muscle cells. Therefore, receptors that otherwise mediate vasoconstriction in vascular muscle cells are apparently also expressed in the cultured, phenotypically modulated SMCs. However, cultured SMCs and cells in vascular muscle seem to differ in the mechanism of the Ca2+ response by α2- adrenoceptors and NPY receptors. It is possible that these changes are typical of proliferating SMCs, which are increasingly found in arteriosclerotic vessels under pathophysiological conditions.</p> |
| title | Characterization of Adrenoceptors in the Vessel Wall and in Cultured Vascular Wall Cells of the Porcine Aorta |
| topic | Porcine Aorta Adrenoreceptors Endothelial Cells Smooth Muscle Cells Valcular Tone Prostaglandins |
| url | https://doi.org/10.5281/zenodo.14697832 |