Vasoactivity of Rucaparib, a PARP-1 Inhibitor, is a Complex Process that Involves Myosin Light Chain Kinase, P2 Receptors, and PARP Itself

Therapeutic inhibition of poly(ADP-ribose) polymerase (PARP), as monotherapy or to supplement the potencies of other agents, is a promising strategy in cancer treatment. We previously reported that the first PARP inhibitor to enter clinical trial, rucaparib (AG014699), induced vasodilation in vivo in xenografts, potentiating response to temozolomide. We now report that rucaparib inhibits the activity of the muscle contraction mediator myosin light chain kinase (MLCK) 10-fold more potently than its commercially available inhibitor ML-9. Moreover, rucaparib produces additive relaxation above the maximal degree achievable with ML-9, suggesting that MLCK inhibition is not solely responsible for dilation. Inhibition of nitric oxide synthesis using L-NMMA also failed to impact rucaparib's activity. Rucaparib contains the nicotinamide pharmacophore, suggesting it may inhibit other NAD+-dependent processes. NAD+ exerts P2 purinergic receptor-dependent inhibition of smooth muscle contraction. Indiscriminate blockade of the P2 purinergic receptors with suramin abrogated rucaparib-induced vasodilation in rat arterial tissue without affecting ML-9-evoked dilation, although the specific receptor subtypes responsible have not been unequivocally identified. Furthermore, dorsal window chamber and real time tumor vessel perfusion analyses in PARP-1-/- mice indicate a potential role for PARP in dilation of tumor-recruited vessels. Finally, rucaparib provoked relaxation in 70% of patient-derived tumor-associated vessels. These data provide tantalising evidence of the complexity of the mechanism underlying rucaparib-mediated vasodilation.

Vasodilator-Stimulated Phosphoprotein (VASP)-dependent and -independent pathways regulate thrombin-induced activation of Rap1b in platelets

Background: Vasodilator-Stimulated Phosphoprotein (VASP) is involved in the inhibition of agonist-induced platelet aggregation by cyclic nucleotides and the adhesion of platelets to the vascular wall. αIIbβ3 is the main integrin responsible for platelet activation and Rap1b plays a key role in integrin signalling. We investigated whether VASP is involved in the regulation of Rap1b in platelets since VASP-null platelets exhibit augmented adhesion to endothelial cells in vivo.

Methods: Washed platelets from wild type and VASP-deficient mice were stimulated with thrombin, the purinergic receptors agonist ADP, or the thromboxane A2 receptor agonist U46619 and Rap1b activation was measured using the GST-RalGDS-RBD binding assay. Interaction of VASP and Crkl was investigated by co-immunoprecipitation, confocal microscopy, and pull-down assays using Crkl domains expressed as GST-fusion proteins.

Results: Surprisingly, we found that activation of Rap1b in response to thrombin, ADP, or U46619 was significantly reduced in platelets from VASP-null mice compared to platelets from wild type mice. However, inhibition of thrombin-induced activation of Rap1b by nitric oxide was similar in platelets from wild type and VASP-null mice indicating that the NO/cGMP/PKG pathway controls inhibition of Rap1b independently from VASP. To understand how VASP regulated Rap1b, we investigated association between VASP and the Crk-like protein (Crkl), an adapter protein which activates the Rap1b guanine nucleotide exchange factor C3G. We demonstrated the formation of a Crkl/VASP complex by showing that: 1) Crkl co-immunoprecipitated VASP from platelet lysates; 2) Crkl and VASP dynamically co-localized at actin-rich protrusions reminiscent of focal adhesions, filopodia, and lamellipodia upon platelet spreading on fibronectin; 3) recombinant VASP bound directly to the N-terminal SH3 domain of Crkl; 4) PKA-mediated VASP phosphorylation on Ser157 abrogated the binding of Crkl.

Conclusions: We identified Crkl as a novel protein interacting with VASP in platelets. We propose that the C3G/Crkl/VASP complex plays a role in the regulation of Rap1b and this explains, at least in part, the reduced agonist-induced activation of Rap1b in VASP-null platelets. In addition, the fact that PKA-dependent VASP phosphorylation abrogated its interaction with Crkl may provide, at least in part, a rationale for the PKA-dependent inhibition of Rap1b and platelet aggregation.

Adventures in the pharmacological analysis of P2 receptors

The pharmacological classification of P2 receptors owes its origin to the pioneering efforts of Geoff Burnstock and those who followed him, research that was conducted primarily in physiological experimental systems. Over recent years, the techniques of molecular biology have been increasingly applied in the study of P2 receptors while, at the same time, advances in their pharmacological analysis have been limited by a lack of potent and selective agonist or antagonist ligands. This has resulted in a classification scheme which is largely structural in nature, with relatively little contribution from pharmacology. Our endeavours in this area have been directed towards the discovery of ligands with which the pharmacological analysis and definition of P2 receptors could be advanced, the ultimate goal being the design of therapeutic agents. This article will describe some of our experiences in this challenging but rewarding Nea. (C) 2000 Elsevier Science B.V. All rights reserved.

ADP ribose is an endogenous ligand for the purinergic P2Y1 receptor

The mechanism by which extracellular ADP ribose (ADPr) increases intracellular free Ca2+ concentration ([Ca2+](i)) remains unknown. We measured [Ca2+](i) changes in fura-2 loaded rat insulinoma INS-1E cells, and in primary beta-cells from rat and human. A phosphonate analogue of ADPr (PADPr) and 8-Bromo-ADPr (8Br-ADPr) were synthesized. ADPr increased [Ca2+](i) in the form of a peak followed by a plateau dependent on extracellular Ca2+. NAD(+), cADPr, PADPr, 8Br-ADPr or breakdown products of ADPr did not increase [Ca2+](i). The ADPr-induced [Ca2+](i) increase was not affected by inhibitors of TRPM2, but was abolished by thapsigargin and inhibited when phospholipase C and IP3 receptors were inhibited. MRS 2179 and MRS 2279, specific inhibitors of the purinergic receptor P2Y1, completely blocked the ADPrinduced [Ca2+](i) increase. ADPr increased [Ca2+](i) in transfected human astrocytoma cells (1321N1) that express human P2Y1 receptors, but not in untransfected astrocytoma cells. We conclude that ADPr is a specific agonist of P2Y1 receptors. (c) 2010 Elsevier Ireland Ltd. All rights reserved.

Inositol 1,4,5-trisphosphate receptors modulate Ca2+ sparks and Ca2+ store content in vas deferens myocytes.

Spontaneous Ca2+ sparks were observed in fluo 4-loaded myocytes from guinea pig vas deferens with line-scan confocal imaging. They were abolished by ryanodine (100 microM), but the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) blockers 2-aminoethoxydiphenyl borate (2-APB; 100 microM) and intracellular heparin (5 mg/ml) increased spark frequency, rise time, duration, and spread. Very prolonged Ca2+ release events were also observed in approximately 20% of cells treated with IP3R blockers but not under control conditions. 2-APB and heparin abolished norepinephrine (10 microM; 0 Ca2+)-evoked Ca2+ transients but increased caffeine (10 mM; 0 Ca2+) transients in fura 2-loaded myocytes. Transients evoked by ionomycin (25 microM; 0 Ca2+) were also enhanced by 2-APB. Ca2+ sparks and transients evoked by norepinephrine and caffeine were abolished by thimerosal (100 microM), which sensitizes the IP3R to IP3. In cells voltage clamped at -40 mV, spontaneous transient outward currents (STOCs) were increased in frequency, amplitude, and duration in the presence of 2-APB. These data are consistent with a model in which the Ca2+ store content in smooth muscle is limited by tonic release of Ca2+ via an IP3-dependent pathway. Blockade of IP3Rs elevates sarcoplasmic reticulum store content, promoting Ca2+ sparks and STOC activity.

Carbachol triggers RyR-dependent Ca(2+) release via activation of IP(3) receptors in isolated rat gastric myocytes.

Possible interactions between different intracellular Ca(2+) release channels were studied in isolated rat gastric myocytes using agonist-evoked Ca(2+) signals. Spontaneous, local Ca(2+) transients were observed in fluo-4-loaded cells with linescan confocal imaging. These were blocked by ryanodine (100 microM) but not by the inositol 1,4,5-trisphosphate receptor (IP(3)R) blocker, 2-aminoethoxydiphenyl borate (100 microM), identifying them as Ca(2+) sparks. Caffeine (10 mM) and carbachol (10 microM) initiated Ca(2+) release at sites which co-localized with each other and with any Ca(2+) spark sites. In fura-2-loaded cells extracellular 2-aminoethoxydiphenyl borate and intracellular heparin (5 mg ml(-1)) both inhibited the global cytoplasmic [Ca(2+)] transient evoked by carbachol, confirming that it was IP(3)R-dependent. 2-Aminoethoxydiphenyl borate and heparin also increased the response to caffeine. This probably reflected an increased Ca(2+) store content since 2-aminoethoxydiphenyl borate more than doubled the amplitude of transients evoked by ionomycin. Ryanodine completely abolished carbachol and caffeine responses but only reduced ionomycin transients by 30 %, suggesting that blockade of carbachol transients by ryanodine was not simply due to store depletion. Double labelling of IP(3)Rs and RyRs demonstrated extensive overlap in their distribution. These results suggest that carbachol stimulates Ca(2+) release through co-operation between IP(3)Rs and RyRs, and implicate IP(3)Rs in the regulation of Ca(2+) store content.

Roles of norepinephrine and ATP in sympathetically evoked vasoconstriction in rat tail and hindlimb in vivo.

In anesthetized rats, we characterized the contributions of norepinephrine (NE) and ATP to changes in tail and hindlimb (femoral) vascular resistances (TVR and FVR, respectively) evoked by three patterns of sympathetic stimulation: 1) couplets (2 impulses at 20 Hz), 2) short trains (20 impulses at 20 Hz), and 3) a natural irregular pattern previously recorded from a sympathetic fiber innervating the rat tail artery. All stimuli evoked greater changes in TVR than FVR. Judging from the effects of the -adrenoceptor antagonist phentolamine, the purinergic receptor antagonist suramin, or ,-methylene ATP (which desensitizes P2X receptors), we propose that NE has a major role in the constriction evoked by the couplet, as well as by the short train and by the low- and high-frequency components of the natural pattern, but that considerable synergy occurred between the actions of ATP and NE. This contrasts with previous in vitro studies that indicated that ATP dominates vascular responses evoked by sympathetic stimulation with a few impulses at low frequency and that NE dominates responses to longer trains or at high frequencies.

Simultaneously multiply-configurable or superposed molecular logic systems composed of ICT (internal charge transfer) chromophores and fluorophores integrated with one- or two-ion receptors

Integrated "ICT chromophore-receptor" systems show ion-induced shifts in their electronic absorption spectra. The wavelength of observation can be used to reversibly configure the system to any of the four logic operations permissible with a single input (YES, NOT, PASS 1, PASS 0), under conditions of ion input and transmittance output. We demonstrate these with dyes integrated into Tsien's calcium receptor, 1-2. Applying multiple ion inputs to 1-2 also allows us to perform two- or three-input OR or NOR operations. The weak fluorescence output of 1 also shows YES or NOT logic depending on how it is configured by excitation and emission wavelengths. Integrated "receptor(1)-ICT chromophore-receptor(2)" systems 3-5 selectively target two ions into the receptor terminals. The ion-induced transmittance output of 3-5 can also be configured via wavelength to illustrate several logic types including, most importantly, XOR. The opposite effects of the two ions on the energy of the chromophore excited state is responsible for this behaviour. INHIBIT and REVERSE IMPLICATION are two of the other logic types seen here. Integration of XOR logic with a preceding OR operation can be arranged by using three ion inputs. The fluorescence output of these systems can be configured via wavelength to display INHIBIT or NOR logic under two-input conditions. The superposition or multiplicity of logic gate configurations is an unusual consequence of the ability to simultaneously observe multiple wavelengths.