John R. Purkiss

The regulation of aortic endothelial cells by purines and pyrimidines involves co‐existing P2y‐purinoceptors and nucleotide receptors linked to phospholipase C

1 We have examined the phospholipase C responses in bovine aortic endothelial cells to purines (ATP, ADP and analogues) and the pyrimidine, uridine triphosphate (UTP). 2 The cells responded to purines in a manner consistent with the presence of P2y purinoceptors; both 2‐methylthioadenosine 5′‐triphosphate (2MeSATP) and adenosine 5′‐0‐(2‐thiodiphosphate) (ADPβS) were potent agonists (EC50 0.41 μm and 0.85 μm respectively) while β, γ‐methylene ATP at 300 μm was not. 3 The cells also responded to UTP. The maximal response to UTP was less than that for either 2MeSATP and ADPβS while adenosine 5′‐0‐(3‐thiotriphosphate) (ATPγS) gave the largest maximal response. 4 The concentration‐effect curve to UTP was additive in the presence of either 2MeSATP or ADPβS. However, the concentration‐effect curves to ATP7S reached the same maximum in the presence or absence of UTP. 5 Suramin, at concentrations between 10 μm and 100 μm was a competitive antagonist for the response to ADPβS and 2MeSATP but not the response to UTP. 6 The results show that there are two separate, co‐existing, receptor populations: P2y‐purinoceptors (responding to purines) and nucleotide receptors (responding to both purines and pyrimidines). We conclude that purines such as ATP/ADP may regulate aortic endothelial cells by interacting with two phospholipase C‐linked receptors.

Differential regulation of inositol 1,4,5-trisphosphate by co-existing P2Y-purinoceptors and nucleotide receptors on bovine aortic endothelial cells

1 We have examined the inositol 1,4,5‐trisphosphate (Ins(1,4,5)P3) responses in bovine aortic endothelial (BAE) cells to purines (ATP, ADP and analogues) and the pyrimidine, uridine triphosphate (UTP). 2 Exchange of medium on BAE cells in the absence of agonist was found to be a stimulus for Ins(1,4,5)P3 generation. BAE cells stimulated with 100 μm ATP, 30 μm 2MeSATP (an agonist at P2Y‐purinoceptors but not nucleotide receptors) or 100 μm UTP (an agonist at nucleotide receptors but not P2Y‐purinoceptors) gave Ins(1,4,5)P3 responses above that caused by exchange of medium. The time course was rapid, with peak response within the first 5 s and levels returning close to basal after 30 s of stimulation. 3 Significant differences in Ins(1,4,5)P3 responses to 100 μm UTP and 30 μm 2MeSATP stimulation were observed. The response to UTP was reproducibly more sustained than that to 2MeSATP. 4 Stimulation of BAE cells with 100 μm UTP plus 30 μm 2MeSATP produced a response statistically indistinguishable from that predicted by addition of the responses to the two agonists in isolation. 5 The Ins(1,4,5)P3 response to UTP was attenuated to 25% of control by pretreatment of BAE cells with pertussis toxin. Responses to 2MeSATP and ADP were essentially unaffected. ATP stimulation was reduced to 65% of control. 6 Activation of protein kinase C with tetradecanoyl phorbol acetate (TPA) profoundly inhibited Ins(1,4,5)P3 responses to 2MeSATP and ADP but had no effect on UTP stimulation. The protein kinase C inhibitor, Ro 31–8220, enhanced responses to 2MeSATP, ADP and ATP but no effect was observed on UTP stimulation. 7 These observations show that nucleotide and P2Y‐receptors mobilise the second messenger Ins (1,4,5)P3 by separate routes resulting in different patterns of generation and suggest that while ATP activates both receptors, ADP principally influences these cells by interacting with the P2Y‐purinoceptors.

Mobilization of Inositol 1,4,5‐Trisphosphate‐Sensitive Ca2+ Stores Supports Bradykinin‐ and Muscarinic‐Evoked Release of [3H]Noradrenaline from SH‐SY5Y Cells

Abstract: The human neuroblastoma cell line SH‐SY5Y, maintained at confluence for 14 days, released [3H]‐noradrenaline ([3H]NA) when stimulated with either the muscarinic receptor agonist methacholine or bradykinin. The major fraction of release was rapid, occurring in <10 s, whereas nicotine‐evoked release was slower. When the extracellular [Ca2+] ([Ca2+]e) was buffered to ∼50–100 nM, release evoked by nicotine was abolished, whereas that in response to methacholine or bradykinin was reduced by ∼50% with EC50 values of −5.46 ± 0.05 M and −7.46 ± 0.06 M (log10), respectively. Methacholine and bradykinin also produced rapid elevations of both inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3] and intracellular free [Ca2+] ([Ca2+]i). These elevations were reduced at low [Ca2+]e and under these conditions the EC50 values for peak elevation of [Ca2+]i were −6.00 ± 0.14 M for methacholine and −7.95 ± 0.34 M for bradykinin (n = 3 for all EC50 determinations). At low [Ca2+]e, depletion of nonmitochondrial intracellular Ca2+ stores with the Ca2+‐ATPase inhibitor thapsigargin produced a transient small elevation of [Ca2+]i and a minor release of [3H]NA. At low [Ca2+]e, thapsigargin abolished elevation of [Ca2+]i in response to methacholine and bradykinin and completely inhibited their stimulation of [3H]NA release. It is proposed, therefore, that Ca2+ release from Ins(1,4,5)P3‐sensitive stores is a major trigger of methacholine‐ and bradykinin‐evoked [3H]NA release in SH‐SY5Y cells.

Endothelin‐1 stimulated phospholipase D in A10 vascular smooth muscle derived cells is dependent on tyrosine kinase Evidence for involvement in stimulation of mitogenesis

The mechanism whereby endothelin stimulates motogenesis of vascular smooth muscle cells is not understood. Here we show that endothelin‐1 stimulates phospholipase D by a protein kinase C and tyrosine kinase dependent mechanism, and present evidence that implicate the phosphatidic acid formed by phospholipase D in the mitogenic response.

Lack of Phospholipase D Activity in Chromaffin Cells: Bradykinin-Stimulated Phosphatidic Acid Formation Involves Phospholipase C in Chromaffin Cells but Phospholipase D in PC12 Cells

The role of lipid‐bound second messengers in the regulation of neurotransmitter secretion is an important but poorly understood subject. Both bovine adrenal Chromaffin cells and rat phoeochromocytoma (PC12) cells, two widely studied models of neuronal function, respond to bradykinin by generating phosphatidic acid (PA). This putative second messenger may be produced by two receptor‐linked pathways: sequential action of phospholipase C (PLC) and diacylglycerol kinase(DAG kinase), or directly by phospholipase D (PLD). Here we show that bradykinin stimulation of Chromaffin cells prelabelled (24 h) with 32P1 leads to production of [32P]PA which is not affected by 50 mM butanol. However, bradykinin stimulation of PC12 cells leads to [32P]PA formation, all of which is converted to phosphatidylbutanol in the presence of butanol. When Chromaffin cells prelabelled with [3H]choline were stimulated with bradykinin there was no enhancement of formation of water soluble products of phosphatidylcholine hydrolysis. When chromaffin cells were permeabilised with pneumolysin and incubated in the presence of [γ‐32P]ATP, the formation of [32P]PA was still stimulated by bradykinin. These results show that, although both neuronal models synthesize PA in response to bradykinin, they do so by quite different routes: PLC/DAG kinase for chromaffin cells and PLD for PC12 cells. The observation that neither bradykinin nor tetradecanoyl phorbol acetate stimulate PLD in chromaffin cells suggests that these cells lack PLD activity. The conservation of PA formation, albeit by different routes, may indicate an essential role of PA in the regulation of cellular events by bradykinin.

Evidence for a nucleotide receptor on adrenal medullary endothelial cells linked to phospholipase C and phospholipase D

1 We have investigated whether the ‘atypical’ P2‐purinoceptor previously described on adrenal microvasculature endothelial cells is a nucleotide receptor (responds to pyrimidines and purines) and is linked to phospholipase D as well as phospholipase C. 2 Cultured bovine adrenal medullary endothelial (BAME) cells responded to the pyrimidine UTP, as well as the purines. The total [3H]‐inositol phosphate responses were with a rank order of UTP > ATP‐ = adenosine 5′‐O‐(3‐thio‐triphosphate) (ATPγS) >> 2MeSATP. The selective P2x agonist β, γ‐methylene ATP was inactive. 3 Construction of dose‐response curves to ATP, ATPγS and UTP in the presence and absence of additional agonists showed that responses to ATPγS and UTP were not additive, nor were those to UTP and ATP. This suggests that purines and pyrimidines acted via a common nucleotide receptor. 4 32P‐labelled BAME cells, in the presence of butanol, produced [32P]‐phosphatidylbutanol (PBut) when stimulated with ATPγS or the protein kinase C activator, tetradecanoyl phorbol acetate (TPA). 5 Cells labelled with [3H]‐palmitate and stimulated in the presence of butanol generated [3H]‐PBut with the same order of agonist potencies seen for inositol phosphate responses. 6 The protein kinase C inhibitor, Ro 31–8220, abolished TPA and agonist stimulation of [3H]‐PBut production. 7 These observations, and our related studies on bovine aortic endothelial cells, provide the first demonstration of a phospholipase C linked nucleotide receptor on vascular endothelial cells. It is concluded that BAME cells express a nucleotide receptor linked to phospholipase C and phospholipase D, but that activation of phospholipase D is probably down‐stream of phospholipase C.

Stimulation of phospholipase C in cultured microvascular endothelial cells from human frontal lobe by histamine, endothelin and purinoceptor agonists

1 Cultures of endothelial cells derived from the microvasculature of human frontal lobe have been investigated for phospholipase C (PLC) responses to histamine, endothelins and purinoceptor agonists. 2 Using cells prelabelled with [3H]‐inositol and measuring total [3H]‐inositol (poly)phosphates, histamine acting at H1 receptors stimulated a substantial response with an EC50 of about 10 μm. 3 Endothelin‐1 also gave a clear stimulation of phosphoinositide‐specific phospholipase C. Both concentration‐response curves and binding curves showed effective responses and binding in the rank order of endothelin‐1>sarafotoxin S6b>endothelin‐3, suggesting an ETA receptor. 4 Assay of total [3H]‐inositol (poly)phosphates showed no response to the purinoceptor agonists, 2‐methylthioadenosine 5′‐trisphosphate (2MeSATP), adenosine 5′‐O‐(3‐thiotrisphosphate) (ATPγS) or β,γ‐methylene ATP. Both ATP and UTP gave a small PLC response. 5 Similarly, when formation of [32P]‐phosphatidic acid from cells prelabelled with 32Pi was used as an index of both PLC and phospholipase D, a small response to ATP and UTP was seen but there was no response to the other purinoceptor agonists tested. 6 Study by mass assay of stimulation by ATP of inositol (1,4,5) trisphosphate accumulation revealed a transient response in the first few seconds, a decline to basal, followed by a small sustained response. 7 These results show that human brain endothelial cells in culture are responsive to histamine and endothelins in a manner which may regulate brain capillary permeability. Purines exert a lesser influence.

Stimulation of phosphatidic acid synthesis in bovine aortic endothelial cells in response to activation of P2-purinergic receptors

In this study we used the bovine thoracic aorta endothelial cell line AG 4762 and primary bovine aortic endothelial cells to investigate the formation of phosphatidic acid (PA) in response to activation of P2-purinergic receptors. 2-Methylthio ATP (2MeSATP) stimulated the formation of [32P]-PA in bovine aortic endothelial cells labelled with 32P(i) for 2.5 hr. A comparison of the response to other ATP analogues suggests that this was mediated via a P2Y-purinergic receptor. Using various agonists at 30 microM there was a correlation between the formation of [32P]PA and of total inositol phosphates in the presence of lithium. The 2MeSATP-stimulated accumulation of [32P]PA showed an initial high rate, followed by a more sustained slower rate. The initial response was independent of extracellular calcium while the later response was dependent on calcium influx. The protein kinase C stimulator phorbol myristate acetate (PMA) produced only a very small enhancement of [32P]PA accumulation compared to 2MeSATP. The 2MeSATP stimulation of both inositol phosphates and [32P]PA was almost eliminated by the presence of PMA. Using cells prelabelled with [3H]methylcholine 2MeSATP produced only a small non-significant enhancement of [3H]choline formation; PMA by contrast formed a much larger amount of [3H]choline. There was no evidence of a change in [3H]phosphocholine. The dissociation between phospholipase D (PLD) activation and [32P]PA accumulation and the correlation between stimulation of [32P]PA accumulation and phospholipase C (PLC) activation all suggest that, using this protocol for labelling cells, the principle route of the stimulation of formation of [32P]PA is via the activation of PLC followed by metabolism of diacylglycerol (DAG) by DAG kinase. These results show that activation of P2Y-purinergic receptors on aortic endothelial cells leads to the formation of phosphatidic acid and that both PLD and PLC pathways are likely to contribute to this response.

Ionomycin induced changes in intra-cellular free calcium in SH-SY5Y human neuroblastoma cells: sources of calcium and effects on [3H]-noradrenaline release

In adherent SH-SY5Y human neuroblastoma cells cultured for 14 days to promote uptake and release of [3H]-noradrenaline, ionomycin induced a biphasic elevation of the intracellular [Ca2+] ([Ca2+]i). This consisted of a rapid transient elevation followed by a marked, persistent secondary elevation. Further study indicated that the peak [Ca2+]i elevation was dependent upon intracellular Ca2+ whilst the secondary elevation was dependent upon extracellular Ca2+. This profile of response and dependence upon intracellular and extracellular sources of Ca2+ was similar to that evoked by the muscarinic agonist, methacholine but was independent of inositol 1,4,5-trisphosphate generation. Ionomycin also stimulated the release of [3H]-noradrenaline from preloaded cells. Both intracellular and extracellular sources of Ca2+ were needed for the full response and synergised to effect release. Thus, in adherent SH-SY5Y cells, ionomycin elevates [Ca2+]i in a complex way in a manner partly analogous to the elevation of [Ca2+]i by agonists of phosphoinositidase C-linked receptors. Furthermore the effects of [Ca2+]i elevation on [3H]-noradrenaline release by these two processes are similar. Such functional consequences may, however, differ under circumstances where the profile and source of Ca2+ for ionomycin-mediated changes differs to that of receptor agonists.

Phospholipase D activation regulates endothelin-1 stimulation of phosphoinositide-specific phospholipase C in SK-N-MC cells.

Endothelin‐1 (ET‐1) is known to stimulate phospholipase C (PLC) activity in SK‐N‐MC human neuroblastoma/epithelioma cells: here we show that phospholipase D(PLD) is also stimulated. The generation of inositol 1,4,5‐trisphosphate (Ins(1,4,5)P3) by ET‐1‐stimulated PLC was attenuated by protein kinase C (PKC) activation and enhanced by PKC inhibition. An enhancement of ET‐1‐stimulated Ins(1,4,5)P3 accumulation was also seen when the product of PLD activity was either diverted into phosphatidyl butanol in the presence of butanol, or phosphatidate phosphohydrolase (PPH) activity was inhibited by dl‐propranolol. We conclude that there is an inhibitory, PKC‐mediated, feedback loop in these cells which is dependent, in part, on the activation of PKC by product(s) of the PLD/PPH pathway. This provides a novel role for agonist‐stimulated PLD activation.