David W. Gray

Novel signal transduction pathway mediating endothelium-dependent β-adrenoceptor vasorelaxation in rat thoracic aorta

1 Isoprenaline (3 × 10−8−10−5 m), salbutamol (3 × 10−7−10−4 m) and forskolin (3 × 10−9−3 × 10−7 m) relaxed rat isolated thoracic aortic rings contracted with noradrenaline (10−7 m). Removal of the endothelium from the aortic rings abolished the effect of acetylcholine (10−6 m) and completely prevented the vascular relaxation induced by isoprenaline, salbutamol or forskolin. 2 The isoprenaline concentration‐relaxation curve was shifted in parallel to the right about 10 fold by propranolol (3 × 10−7 m) with no change in the maximum response, showing that the relaxation was mediated by a β‐adrenoceptor. 3 The inhibitor of nitric oxide synthesis, NG‐nitro‐l‐arginine (l‐NOARG; 10−5 m), shifted the isoprenaline relaxation curve to the right and reduced the maximum response. 4 Isoprenaline (10−6 m) relaxed noradrenaline‐induced tone by approximately 95% and at the same time increased levels of adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) 4 fold and guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) 12 fold in the aortic rings. Sodium nitroprusside (3 × 10−8 m) relaxed noradrenaline‐evoked tone by 82% without changing levels of cyclic AMP but raised cyclic GMP 19 fold. 5 Forskolin (10−7 m) relaxed noradrenaline‐induced tone by approximately 41% and, like isoprenaline, increased levels of cyclic AMP (2.5 fold) and cyclic GMP (12 fold) in the aortic rings. 6 Removal of the endothelium abolished the relaxant effects of isoprenaline (10−6 m) and also the associated accumulation of cyclic AMP and cyclic GMP. 7 l‐NOARG (10−5 m) inhibited the relaxant responses and accumulation of cyclic GMP induced by isoprenaline (10−6 m) and forskolin (10−7 m) without affecting the associated cyclic AMP accumulation. 8 It is concluded that, in the rat aorta, isoprenaline acts through a β‐adrenoceptor on the endothelium to raise cyclic AMP and that this may, directly or indirectly, release nitric oxide to evoke vascular relaxation via the increase in cyclic GMP. The importance of this novel transduction pathway for cardiovascular regulation remains to be determined.

Human α‐calcitonin gene‐related peptide stimulates adenylate cyclase and guanylate cyclase and relaxes rat thoracic aorta by releasing nitric oxide

1 The signal transduction pathway for vasorelaxation induced by human α‐calcitonin gene‐related peptide (human α‐CGRP) was studied in rat thoracic aortic rings preconstricted with noradrenaline (10−7 m). 2 Vasorelaxation by human α‐CGRP was inhibited by haemogobin (10−6 m) and methylene blue (10−5 m) but was unaffected by ibuprofen (10−5 m). 3 Acetylcholine caused a 16 fold increase in levels of guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) with levels of adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) being unaltered. Human α‐CGRP caused a 12 fold increase in levels of cyclic GMP but, in contrast to acetylcholine, evoked a 2.5 fold rise in levels of cyclic AMP. The rises in cyclic nucleotides evoked by human α‐CGRP and acetylcholine were dependent on the presence of an intact endothelium. 4 NG‐nitro‐l‐arginine (l‐NOARG: 10−5 m), which inhibits nitric oxide synthase, inhibited the relaxant response to human α‐CGRP and cyclic GMP accumulation without affecting the cyclic AMP accumulation. 5 The data presented in this paper suggests that human α‐CGRP relaxes the rat thoracic aorta by releasing nitric oxide and stimulating guanylate cyclase. The stimulation of adenylate cyclase by human α‐CGRP probably precedes the activation of nitric oxide synthase but could be unrelated to the relaxant response.

Nitric oxide synthesis inhibitors attenuate calcitonin gene-related peptide endothelium-dependent vasorelaxation in rat aorta

The effect of inhibitors of nitric oxide synthesis were examined on the endothelium-dependent relaxation induced by acetylcholine and human alpha-calcitonin gene-related peptide (CGRP) on rat isolated aortic rings preconstricted with noradrenaline. The endothelium-dependent vasorelaxation induced by acetylcholine and CGRP was inhibited by NG-monomethyl-L-arginine (L-NMMA) and NG-nitro-L-arginine (L-NOARG) in a concentration-related fashion. The inhibition of endothelium-dependent relaxation by L-NMMA and L-NOARG was partially reversed by L-arginine but not by D-arginine for both acetylcholine and CGRP. The data presented suggests that CGRP, like acetylcholine; relaxes the rat aorta via the release of nitric oxide.

Modulatory effects of NMDA on phosphoinositide responses evoked by the metabotropic glutamate receptor agonist 1S,3R-ACPD in neonatal rat cerebral cortex.

1 The effect of NMDA‐receptor stimulation on phosphoinositide signalling in response to the metabotropic glutamate receptor agonist 1‐aminocyclopentane‐1S,3R‐dicarboxylic acid (1S,3R‐ACPD) has been examined in neonatal rat cerebral cortex slices. 2 Total [3H]‐inositol phosphate ([3H]‐InsPx) accumulation, in the presence of 5 mm LiCl, in [3H]‐inositol pre‐labelled slices was concentration‐dependently increased by 1S,3R‐ACPD (EC50 16.6 μm) and, at a maximally effective concentration, 1S,3R‐ACPD (300 μm) increased [3H]‐InsPx accumulation by 12.8 fold over basal values. 3 [3H]‐InsPx accumulation stimulated by 1S,1R‐ACPD was enhanced by low concentrations of NMDA (3–30 μm), but not by higher concentrations (> 30 μm). [3H]‐InsPx accumulations stimulated by 1S,3R‐ACPD in the absence or presence of 10 μm NMDA were linear with time, at least over the 15 min period examined; however, in the presence of 100 μm NMDA the initial enhancement of 1S,3R‐ACPD‐stimulated phosphoinositide hydrolysis progressively decreased with time. 4 In the presence of a maximal enhancing concentration of NMDA (10 μm), the response to 1S,3R‐ACPD (300 μm) was increased 1.9 fold and the EC50 for agonist‐stimulated [3H]‐InsPx accumulation decreased about 4 fold. The enhanced response to the metabotropic agonist was concentration‐dependently inhibited by competitive and uncompetitive antagonists of NMDA‐receptor activation. 5 1S,3R‐ACPD also stimulated inositol 1,4,5‐trisphosphate (Ins(1,4,5)P3) mass accumulation with an initial peak response (5–6 fold over basal) at 15 s decaying to a smaller (2 fold), but persistent elevated accumulation (1–10 min). 6 Co‐addition of 10 or 100 μm NMDA enhanced the initial peak Ins(1,4,5)P3 response to 1S,3R‐ACPD. However, the enhancing effect was only maintained over 10 min in the presence of 10 μm NMDA, whilst in contrast, 100 μm NMDA ceased to cause a significant enhancement of the metabotropic response by 5 min and completely suppressed 1S,3R‐ACPD‐stimulated Ins(1,4,5)P3 accumulation at 10 min. 7 Both basal and 1S,3R‐ACPD‐stimulated Ins(1,4,5)P3 accumulations were reduced when slices were incubated in nominally Ca2+‐free medium. Under these conditions only a concentration‐dependent enhancement of the response was observed (EC50 for NMDA facilitation of 1S,3R‐ACPD‐stimulated Ins(1,4,5)P3 accumulation of 32 μm). 8 These experiments have revealed that at low concentrations, NMDA can dramatically potentiate 1S,3R‐ACPD‐stimulated phosphoinositide hydrolysis, probably by a Ca2+‐dependent facilitation of agonist‐stimulated phosphoinositide‐specific phospholipase C activity. Higher concentrations of NMDA result in time‐dependent inhibition of the metabotropic agonist‐stimulated response. We believe the former effect could be fundamental in glutamate receptor ‘cross‐talk’, whereas the latter may reflect a Ca2+‐dependent neurotoxic effect of NMDA on the neonatal cerebral cortex slices.

Receptors mediating CGRP-induced relaxation in the rat isolated thoracic aorta and porcine isolated coronary artery differentiated by hα CGRP8–37

Receptors mediating CGRP‐induced vasorelaxation were investigated in rat thoracic aorta and porcine left anterior descending (LAD) coronary artery and anterior interventricular artery (AIA), using CGRP agonists, homologues and the antagonist hα CGRP8–37. In the endothelium‐intact rat aorta, hα CGRP, hβ CGRP, rat β CGRP and human adrenomedullin caused relaxation with similar potencies. Compared with hα CGRP, rat amylin was about 25 fold less potent, while [Cys(ACM2,7)] hα CGRP and salmon calcitonin were at least 1000 fold weaker. Hα CGRP8–37 (up to 10−5 M) did not antagonize responses to hα CGRP, hβ CGRP or rat β CGRP (apparent pKB <5). Peptidase inhibitors did not increase either the effect of hα CGRP or [Cys(ACM,2,7)] hα CGRP, while hα CGRP8–37 remained inactive. Endothelium‐dependent relaxation produced by hα CGRP was accompanied by increases in cyclic AMP and cyclic GMP, that were not inhibited by hα CGRP8–37 (10−5 M). In porcine LAD and AIA, hα CGRP produced relaxation in an endothelium‐independent manner. Hα CGRP8‐37 competitively antagonized hα CGRP responses (pA2 6.3 and 6.7 (Schild slope 0.9±0.1, each), in LAD and AIA, respectively). In LAD artery, hα CGRP‐induced relaxation was accompanied by increases in cyclic AMP that were inhibited by hα CGRP8–37 (10−7–10−5 M). In conclusion, the antagonist affinity for hα CGRP8–37 in porcine coronary artery is consistent with a CGRP1 receptor, while the lack of hα CGRP8–37 antagonism in rat aorta could suggest either a CGRP receptor different from CGRP1 and CGRP2 type, or a non‐CGRP receptor.

Modulation of muscarinic cholinoceptor-stimulated inositol 1,4,5-trisphosphate accumulation by N-methyl-D-aspartate in neonatal rat cerebral cortex.

The mechanisms by which N-methyl-D-aspartate (NMDA) receptor activation can modulate muscarinic receptor-stimulated phosphoinositide turnover have been studied in neonatal rat cerebral cortex slices. A maximally effective concentration of carbachol (1 mM) caused a large stimulation of both total [3H]inositol phosphate ([3H]InsPx) accumulation (30-40-fold over basal levels after 15 min in the presence of 5 mM LiCl) and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] mass accumulation (consisting of a rapid peak increase of about 8-10-fold within 15 sec followed by a sustained plateau rise of 4-5-fold which persisted for > 10 min). Low concentrations of NMDA enhanced carbachol-stimulated [3H]InsPx and Ins(1,4,5)P3 accumulations with a maximal effect being observed at 10 microM NMDA. However, at higher concentrations of NMDA (30-300 microM) a dramatic inhibition of these indices of phosphoinositide turnover was observed. Time-course studies demonstrated that NMDA (100 microM) caused a significant enhancement of the initial increases in [3H]InsPx and Ins(1,4,5)P3 accumulations stimulated by carbachol, with the profound inhibitory effects becoming evident at longer incubation times. The modulatory effects of NMDA were antagonized by D-2-amino-5-phosphonopentanoate and MK-801. Reducing extracellular calcium concentration ([Ca2+]e) to the low micromolar range decreased basal Ins(1,4,5)P3 accumulation and attenuated the response to carbachol. Under these conditions NMDA (10-100 microM) caused only a potentiation of agonist-stimulated Ins(1,4,5)P3 accumulation. Under control conditions ([Ca2+]e = 1.3 mM), addition of MK-801 (1 microM) 10 min after carbachol + 100 microM NMDA challenge failed to reverse the inhibitory effect of NMDA on carbachol-stimulated [3H]InsPx accumulation. Furthermore, pre-incubation of cerebral cortex slices with 100 microM NMDA for 15 min (followed by extensive washing of slices to remove NMDA) dramatically decreased [3H]inositol incorporation into the cellular inositol phospholipid fraction and decreased basal and carbachol-stimulated Ins(1,4,5)P3 mass accumulations. We conclude that the enhancement of agonist-stimulated phosphoinositide turnover seen at concentrations of NMDA up to 10 microM may be due to Ca2+ entry and Ca2+ facilitation of phosphoinositide-specific phospholipase C activity. In contrast, the inhibitory effect of high concentrations of NMDA on agonist-stimulated phosphoinositide turnover may be due to progressive, irreversible and, at least in part, Ca(2+)-dependent damage to the cell populations in the slice preparation responding to muscarinic-receptor stimulation.

Differential effects of lithium on muscarinic cholinoceptor-stimulated CMP-phosphatidate accumulation in cerebellar granule cells, CHO-M3 cells, and SH-SY5Y neuroblastoma cells.

Abstract: The ability of lithium to potentiate muscarinic cholinoceptor‐stimulated CMP‐phosphatidate (CMP.PA) accumulation has been examined in various cells in which muscarinic cholinoceptor agonists evoke a phosphoinositide response. Cell types examined include rat cerebellar granule cells, Chinese hamster ovary cells transfected to express the human muscarinic M3 receptor (CHO‐M3 cells), and SH‐SY5Y neuroblastoma cells. Neither carbachol (1 mM) nor lithium (10 mM) caused significant increases in CMP.PA accumulation in rat cerebellar granule cells; however, when added together for 20 min a linear 17‐fold increase over basal levels was observed. The increase was dependent on the concentration of carbachol and lithium present, and the effect could be reversed by addition of exogenous myo‐inositol (10 mM). Addition of carbachol alone to CHO‐M3 cells caused a five‐fold increase in CMP.PA accumulation. In the presence of lithium, a 70‐fold increase was observed at 20 min after carbachol plus lithium addition. This latter response was concentration dependent and could be abolished by preincubation in the presence of 10 mM myo‐inositol. In contrast, whereas carbachol elicited a three‐fold increase in CMP.PA accumulation in SH‐SY5Y neuroblastoma cells, which reached a plateau 10 min after agonist addition, the response could neither be augmented by addition of lithium nor inhibited by addition of myo‐inositol. These results emphasise that the ability of lithium to affect agonist‐stimulated CMP.PA accumulation is not simply a function of stimulus strength, but is also crucially dependent on the intracellular concentration of inositol.

Modulation of muscarinic cholinoceptor-stimulated inositol 1,4,5- trisphosphate accumulation by N-methyl-D-aspartate in neonatal rat cerebral cortex

The mechanisms by which N-methyl-D-aspartate (NMDA) receptor activation can modulate muscarinic receptor-stimulated phosphoinositide turnover have been studied in neonatal rat cerebral cortex slices. A maximally effective concentration of carbachol (1 mM) caused a large stimulation of both total [3H]inositol phosphate ([3H]InsPx) accumulation (30-40-fold over basal levels after 15 min in the presence of 5 mM LiCl) and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] mass accumulation (consisting of a rapid peak increase of about 8-10-fold within 15 sec followed by a sustained plateau rise of 4-5-fold which persisted for > 10 min). Low concentrations of NMDA enhanced carbachol-stimulated [3H]InsPx and Ins(1,4,5)P3 accumulations with a maximal effect being observed at 10 microM NMDA. However, at higher concentrations of NMDA (30-300 microM) a dramatic inhibition of these indices of phosphoinositide turnover was observed. Time-course studies demonstrated that NMDA (100 microM) caused a significant enhancement of the initial increases in [3H]InsPx and Ins(1,4,5)P3 accumulations stimulated by carbachol, with the profound inhibitory effects becoming evident at longer incubation times. The modulatory effects of NMDA were antagonized by D-2-amino-5-phosphonopentanoate and MK-801. Reducing extracellular calcium concentration ([Ca2+]e) to the low micromolar range decreased basal Ins(1,4,5)P3 accumulation and attenuated the response to carbachol. Under these conditions NMDA (10-100 microM) caused only a potentiation of agonist-stimulated Ins(1,4,5)P3 accumulation. Under control conditions ([Ca2+]e = 1.3 mM), addition of MK-801 (1 microM) 10 min after carbachol + 100 microM NMDA challenge failed to reverse the inhibitory effect of NMDA on carbachol-stimulated [3H]InsPx accumulation. Furthermore, pre-incubation of cerebral cortex slices with 100 microM NMDA for 15 min (followed by extensive washing of slices to remove NMDA) dramatically decreased [3H]inositol incorporation into the cellular inositol phospholipid fraction and decreased basal and carbachol-stimulated Ins(1,4,5)P3 mass accumulations. We conclude that the enhancement of agonist-stimulated phosphoinositide turnover seen at concentrations of NMDA up to 10 microM may be due to Ca2+ entry and Ca2+ facilitation of phosphoinositide-specific phospholipase C activity. In contrast, the inhibitory effect of high concentrations of NMDA on agonist-stimulated phosphoinositide turnover may be due to progressive, irreversible and, at least in part, Ca(2+)-dependent damage to the cell populations in the slice preparation responding to muscarinic-receptor stimulation.

Muscarinic Cholinoceptor‐Stimulated Synthesis and Degradation of Inositol 1,4,5‐Trisphosphate in the Rat Cerebellar Granule Cell

Abstract: A detailed analysis of the generation and subsequent metabolism of inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3] following muscarinic cholinoceptor stimulation in primary cultures of rat cerebellar granule cells has been undertaken. Following incubation of cerebellar granule cell cultures with [3H]inositol for 48 h, labelling of the inositol phospholipid pool approached equilibrium. Significant basal labelling of inositol pentakisphosphate (InsP5) and inositol hexakisphosphate (InsP6), as well as inositol mono‐ to tetrakisphosphate, fractions was observed. Addition of carbachol (1 mM) caused an immediate increase in level of Ins(1,4,5)P3 (peak increase two‐fold over basal by 60 s), which was well‐maintained over the initial 300 s following agonist addition. In contrast, only a modest, more slowly developing, increase in inositol tetrakisphosphate accumulation was observed, whereas labelling of InsP5 and InsP6 was entirely unaffected by carbachol stimulation. Analysis of the products of Ins(1,4,5)P3 and inositol 1,3,4,5‐tetrakisphosphate metabolism in broken cell preparations strongly suggested that Ins(1,4,5)P3 metabolism occurs predominantly via the inositol polyphosphate 5‐phosphatase route, with metabolism via the Ins(1,4,5)P3 3‐kinase being a relatively minor pathway. In view of the pattern of inositol (poly)phosphate metabolites observed on stimulation of the muscarinic receptor, it seems likely that, over the time course studied, the inositol polyphosphates are derived principally from phosphoinositide‐specific phospholipase C hydrolysis of phosphatidylinositol 4,5‐bisphosphate, although some hydrolysis of phosphatidylinositol 4‐phosphate cannot be excluded.