Iain L. O. Buxton

Involvement of cyclic GMP in non‐adrenergic, non‐cholinergic inhibitory neurotransmission in dog proximal colon

1 Nitric oxide (NO) may serve as a non‐adrenergic, non‐cholinergic (NANC) neurotransmitter released from enteric inhibitory nerves in the gastrointestinal tract. We tested whether guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) may serve as a second messenger in transducing the NO signal into inhibitory junction potentials (i.j.ps) and relaxation in the canine proximal colon. 2 The membrane permeable analogue of cyclic GMP, 8‐bromo cyclic GMP (8‐Br‐cyclic GMP) mimicked the effects of NO by hyperpolarizing cells near the myenteric border of the circular muscle layer and shortening slow waves in cells near the submucosal surface of the circular muscle layer. 8‐Br‐cGMP also inhibited spontaneous phasic contractions. 3 The specific cyclic GMP phosphodiesterase inhibitor, M&B 22948, hyperpolarized cells near the myenteric border and prolonged the duration of i.j.ps. M&B 22948 also inhibited phasic contractile activity. 4 Methylene blue failed to reduce significantly the amplitude and duration of i.j.ps and had variable effects on contractions. 5 Cyclic GMP levels were assayed in unstimulated muscles and in muscles exposed to exogenous NO and electrical field stimulation. Both stimuli hyperpolarized membrane potential, inhibited contractions, and elevated cyclic GMP levels. 6 Treatment of muscles with l‐NG‐nitroarginine methyl ester (l‐NAME) increased spontaneous contractile activity and lowered cyclic GMP levels. The inhibitory effect of M&B 22948 on contractions was greatly reduced after muscles were treated with l‐NAME. 7 These data support the concept that the effects of NANC nerve stimulation and NO (which may be one of the enteric inhibitory transmitters) may be mediated by cyclic GMP.

Functional Compartmentation of Endothelial P2Y Receptor Signaling

Abstract— The presence of multiple receptors for disparate nucleotides on endothelial cells makes it unclear how the endothelium differentiates among these signals. We propose that endothelial P2Y receptors are organized into cholesterol-rich signaling domains, such as caveolae and respond to nucleotide agonists by mobilizing intracellular calcium. Treatment of endothelial cells with 5 mmol/L &bgr;-methyl-cyclodextrin prevents calcium release in response to the nucleotide receptor agonists 2-methylthio-ATP, ATP, ADP, and UTP, but not the kinin receptor agonist bradykinin, suggesting that depletion of membrane cholesterol disrupts signaling at P2Y receptors and that bradykinin receptors are not prelocalized to cholesterol microdomains in these cells. Direct measurement of cholesterol content after &bgr;-methyl-cyclodextrin treatment of aortic rings reveals a concentration-dependent depletion of cholesterol that parallels functional antagonism of P2Y-mediated relaxation. Nucleotide- and bradykinin-mediated relaxation is disrupted by 5 to 15 mmol/L &bgr; -methyl-cyclodextrin treatment or 1 to 10 &mgr;g/mL filipin III in a concentration-dependent fashion. Norepinephrine contracted aorta treated with A23187 relaxes in an endothelium-dependent fashion despite depletion of 84% of membrane-extractable cholesterol. These data indicate that in the basal state, P2Y receptors but not the kinin receptor may be compartmented to cholesterol-dependent signaling domains in guinea pig endothelium and that cholesterol-rich microdomains in these cells can respond to intracellular calcium in an agonist-specific manner. We suggest that the functional organization of cholesterol-rich signaling microdomains allows agonist-specific responses to increases in intracellular calcium and that this property may be a general phenomenon that permits cells to respond disparately to agonists that may signal through common calcium release pathways.

Evidence for a discrete UTP receptor in cardiac endothelial cells

1 We have examined the effects of various purine and pyrimidine nucleotides upon cells cultured from guinea‐pig cardiac endothelium (CEC), and find the P2Y‐agonist 2‐methylthioadenosine triphosphate (2MeSATP) to be a potent (EC50 = 85±10.2 nM) stimulator of increases in intracellular calcium concentrations, while uridine 5′‐triphosphate (UTP) and adenosine 5′‐triphosphate (ATP) are less potent but equipotent with one another (EC50s = 2.1±0.3 and 1.8±0.2 μm, respectively). 2 While the P2Y receptor exhibited rapid homologous desensitization, this had no effect upon subsequent responsiveness of CEC to either ATP or UTP. Effects of maximal concentrations of ATP and UTP were not only additive, but did not cross‐desensitize. Responses to UTP (but not to ATP or 2MeSATP) were blocked by treatment with pertussis toxin (PTX); all three nucleotides appeared to liberate calcium from an intracellular pool. 3 Suramin (30 μm) significantly (P<0.05) increased the EC50 for ATP‐dependent increases in intracellular calcium (5.3±2.2 μm vs. 2.0±0.9 μm in the absence of suramin), while it completely blocked the response to 2MeSATP. Suramin had no effect upon responses to UTP at concentrations of 100 μm. 4 We conclude that in addition to the P2Y and P2U subtypes of the ATP receptor, an additional receptor responsive to UTP but exhibiting no affinity for purine nucleotides is present in CEC; this ‘pyrimidine receptor’ liberates intracellular calcium via a G‐protein, and may partly mediate the contractile response to UTP in the coronary vasculature.

NO‐induced relaxation of labouring and non‐labouring human myometrium is not mediated by cyclic GMP

In myometrial strips from near‐term non‐labouring human uterus, addition of oxytocin (OT) evoked dose‐dependent (10 – 3000 nM) phasic contractions that were antagonized by atosiban (1 μM) and relaxed by addition of the nitric oxide donor S‐nitroso L‐cysteine (Cys‐NO). In near‐term labouring myometrium, however, addition of OT was ineffective at raising additional tone. In both labouring and non‐labouring tissue, Cys‐NO mediated relaxation of spontaneous or OT‐induced contractions (IC50=1 μM) was unaffected by prior addition of the guanylyl cyclase (GC) inhibitors ODQ (1H‐[1,2,4]oxadiazolo[4,3,‐α]quinoxalin‐1‐one; 1 μM), or methylene blue (MB; 10 μM). Elevation of intracellular cyclic GMP accompanying 30 μM Cys‐NO addition in non‐labouring tissue (7.5 fold) or in labouring tissues (2.5 fold) was completely blocked in tissues that had been pre‐treated with ODQ or MB. Charybdotoxin (ChTx), iberiotoxin (IbTx) and kaliotoxin (KalTx) all shifted the Cys‐NO inhibition curve to the right and reduced the degree of relaxation produced by maximal Cys‐NO treatment (100 μM in non‐labouring tissue; in labouring tissue, KalTx prevented Cys‐NO mediated relaxation in both stimulated and unstimulated tissue. Addition of the NO‐donor S‐nitroso N‐acetyl penicillamine (SNAP) produced a dose‐dependent relaxation of pregnant myometrium while 3‐morpholinosyndonimine (SIN‐1) did not. The failure of SIN‐1 to relax OT‐induced contractions was not due to a failure of the donor to stimulate myometrial GC. We demonstrate that despite the ability of NO to stimulate myometrial GC in pregnant uterine muscle, relaxations are independent of cyclic GMP action. Effects of K+‐channel inhibitors suggests that NO‐induced relaxation in human uterine smooth muscle may be subserved by direct or indirect activation of one or more calcium‐activated K+‐channels.

ATP as a Cotransmittera

The notion that one neuron synthesizes, stores, and releases only one transmitter has been seriously questioned recently.’ The possible existence of cotransmitters in postganglionic sympathetic and parasympathetic neurons has been of particular research interest of late, and much credit for focusing attention on this issue goes to G. Burnstock, who published a commentary in Neuroscience in 1976 entitled “Do Some Nerve Cells Release More Than One Transmitter?”’ Although a number of substances have been proposed to act as cotransmitters with the “classical” monoamine or amino acid transmitters,’ the evidence to date is strongest for a nucleotide, most probably ATP. There has been, naturally enough, a general reluctance in accepting the notion that ATP, a compound that plays an essential role in intracellular function, could be released extracellularly. Nevertheless, the evidence is now abundant that adenine nucleotides and nucleosides do appear extracellularly upon stimulation of nerves, and this, together with the knowledge that these compounds exert a variety of pharmacological actions, makes it reasonable to postulate roles as neuromodulators and neurotransmitters. The tissue for which the data is probably the most extensive in support of a cotransmitter role for ATP is the sympathetically innervated vas deferens. In this paper, we will begin by reviewing the evidence from our laboratory and the laboratories of others that norepinephrine ( N E ) and ATP are cotransmitters in the vas deferens. We will then discuss other neuroeffector junctions, especially vascular smooth muscle, from the standpoint of cotransmission. We will also consider the potential sites of release of endogenous ATP upon transmural nerve stimulation of blood vessels.

Purinergic regulation of vascular endothelial growth factor signaling in angiogenesis.

P2Y purine nucleotide receptors (P2YRs) promote endothelial cell tubulogenesis through breast cancer cell-secreted nucleoside diphosphate kinase (NDPK). We tested the hypothesis that activated P2Y1 receptors transactivate vascular endothelial growth factor receptor (VEGFR-2) in angiogenic signaling. P2Y1R stimulation (10 μM 2-methyl-thio-ATP (2MS-ATP)) of angiogenesis is suppressed by the VEGFR-2 tyrosine kinase inhibitor, SU1498 (1 μM). Phosphorylation of VEGFR-2 by 0.0262 or 2.62 nM VEGF was comparable with 0.01 or 10 μM 2MS-ATP stimulation of the P2Y1R. 2MS-ATP, and VEGF stimulation increased tyrosine phosphorylation at tyr1175. 2MS-ATP (0.1–10 μM) also stimulated EC tubulogenesis in a dose-dependent manner. The addition of sub-maximal VEGF (70 pM) in the presence of increasing concentrations of 2MS-ATP yielded additive effects at 2MS-ATP concentrations <3 μM, whereas producing saturated and less than additive effects at ⩾3 μM. We propose that the VEGF receptor can be activated in the absence of VEGF, and that the P2YR–VEGFR2 interaction and resulting signal transduction is a critical determinant of vascular homoeostasis and tumour-mediated angiogenesis.

Cyclic GMP‐independent effects of nitric oxide on guinea‐pig uterine contractility

1 The role of nitric oxide (NO) in the regulation of uterine contractility has yet to be clearly defined. We evaluated the effect of NO (in the form of S‐nitroso‐L‐cysteine, CysNO) upon uterine contractility and guanosine 3′,5′‐cyclic monophosphate (cyclic GMP) accumulation in pregnant and nonpregnant guinea‐pig myometrium. 2 While CysNO had no effect upon spontaneous contractile activity in either pregnant or nonpregnant uterine tissues, addition of CysNO resulted in an immediate and reversible relaxation of oxytocin‐ or acetylcholine (ACh)‐evoked contractions. 3 Relaxation of agonist‐evoked contractions in response to CysNO was associated with significant elevations in intracellular cyclic GMP concentrations ([cyclic GMP]i). 4 Elevations in [cyclic GMP]i were not required for relaxation, as inhibition of guanylyl cyclase by methylene blue prevented [cyclic GMP]i accumulation while having no effect upon the ability of CysNO to relax agonist‐evoked contractions. 5 Addition of the cyclic GMP‐analogues, 8‐Br‐cyclic GMP and PET‐cyclic GMP, only at high concentrations, produced partial relaxation of agonist‐contracted tissues, suggesting the possibility that cyclic GMP may be sufficient but not necessary for myometrial relaxation. 6 Our studies not only provide evidence for a functional role for NO‐modulation of agonist‐evoked contractions in the pregnant and nonpregnant guinea‐pig uterus, but also that these occur by a mechanism which is not dependent upon guanylyl cyclase activity.

Purinergic regulation of angiogenesis by human breast carcinoma-secreted nucleoside diphosphate kinase

MDA-MB-435S human breast cancer cells (435S) secrete nucleoside diphosphate kinase (NDPK) that supports metastases and is inhibited by epigallocatechin gallate (EGCG) and ellagic acid (EA). We hypothesise that 435S cell-secreted NDPK-B supports tumour formation by modulating ATP levels locally to activate endothelial cell (EC) P2Y receptor-mediated angiogenesis. Epigallocatechin gallate (IC50=8–10 μM) and EA (IC50=2–3 μM) suppressed 435S cell growth, but had less effect on human CD31+ EC growth. Epigallocatechin gallate (IC50=11 μM) and EA (IC50=1 μM) also prevented CD31+ EC tubulogenesis on Matrigel™. 435S cell-conditioned media induced tubulogenesis in a cell number, time, and nucleotide-dependent manner. Ellagic acid (1 μM), but not equimolar EGCG, reduced cell number-dependent angiogenesis. P2Y1 receptor activation by NDPK-generated nucleotide (100 μM ATP) or by 10 μM 2-methyl-thio-ATP (2MS-ATP) promoted tubulogenesis on collagen and was blocked by the P2Y1 antagonist MRS2179 (10 μM). Physiological amounts of purified as well as 435S cell-secreted NDPK also promoted angiogenesis that was attenuated by NDPK depletion or 10 μM MRS2179, indicating a P2Y1 receptor-mediated pathway. These results support the notion that secreted NDPK mediates angiogenesis via P2Y receptor signalling and suggests that novel inhibitors of NDPK may be useful as therapeutics.

Nitric oxide regulation of monkey myometrial contractility

We evaluated the effect of the nitric oxide (NO) donor CysNO (S‐nitroso‐L‐cysteine) and endogenous NO upon spontaneous contractility in non‐pregnant cynomolgus monkeys. We also assessed the role of intracellular guanosine 3′,5′‐cyclic monophosphate ([cyclic GMP]i) as a second messenger for NO in monkey uterine smooth muscle. CysNO reduced spontaneous contractility by 84% (P<0.05) at maximal concentrations, and significantly elevated [cyclic GMP]i (P<0.05). However, increases in [cyclic GMP]i were not required for CysNO‐induced relaxations; CysNO inhibited contractile activity despite the complete inhibition of guanylyl cyclase by methylene blue or LY83,583. Analogues of cyclic GMP had no significant effect upon spontaneous contractile activity. L‐arginine produced a 62% reduction in spontaneous activity (P<0.05) while D‐arginine had no effect. The competitive nitric oxide synthase (NOS) inhibitor Nω‐nitro‐L‐arginine (L‐NOARG) not only blocked L‐arginine‐induced relaxations, but also significantly increased spontaneous contractile activity when added alone (P<0.05); the inactive D‐enantiomer of NOARG had no such effect. While both endogenous NO and the NO donor CysNO relax monkey myometrium, this effect is not causally related to CysNO‐induced elevations in [cyclic GMP]i. The failure of cyclic GMP analogues to alter monkey uterine smooth muscle tension also argues against a role for [cyclic GMP]i in the regulation of uterine contractility. Not only do these findings argue for the existence of a functionally‐relevant NOS in the monkey uterus, but increases in contractile activity seen in the presence of NOS inhibitors suggest a role for NO in the moment‐to‐moment regulation of contractile activity in this organ.

Nucleotide-mediated relaxation in guinea-pig aorta: selective inhibition by MRS2179

The vasodilatory effects of nucleotides in the guinea‐pig thoracic aorta were examined to determine the relationship between molecular expression and function of P2Y receptors. In aortic rings precontracted with norepinephrine, vasodilatory responses to purine nucleotides exhibited a rank‐order of potency of 2‐methylthio‐ATP>ADP>ATP. Responses to UTP, but not UDP suggested a functional role for P2Y4 but not P2Y6 receptors. Aortic endothelial cells express at least four P2Y receptors; P2Y1, P2Y2, P2Y4 and P2Y6. In primary culture, these cells exhibit desensitizing transient calcium responses characteristic of P2Y1, P2Y2 and P2Y4, but not P2Y6 receptors. UDP had no effect on endothelial cell calcium. The pyrimidinergic receptor agonist UTP is capable of eliciting robust vasodilation in aortic rings and causing calcium responses in cultured guineapig aortic endothelial cells. These responses are equivalent to the maximum responses observed to ATP and ADP. Measurement of intracellular calcium release in response to ATP and 2‐methylthio‐ATP were similar, however only the 2‐methylthio‐ATP response was sensitive to the P2Y1 antagonist N6‐methyl‐2′‐deoxyadenosine‐3′,5′‐bisphosphate (MRS2179). In aortic rings, vasodilatory responses to 2‐methylthio‐ATP, ATP and ADP were all blocked by pre‐incubation of tissues with MRS2179. MRS2179 pretreatment had no effect of the ability of UTP to cause relaxation of norepinephrine responses in aortic rings or the ability of UTP to cause calcium release in aortic endothelial cells. We demonstrate robust effects of purine and pyrimidine nucleotides in guineapig aorta and provide functional and biochemical evidence that MRS2179 is a selective P2Y1 antagonist.