James A. Angus

PI 3-kinase p110β : a new target for antithrombotic therapy.

Platelet activation at sites of vascular injury is essential for the arrest of bleeding; however, excessive platelet accumulation at regions of atherosclerotic plaque rupture can result in the development of arterial thrombi, precipitating diseases such as acute myocardial infarction and ischemic stroke. Rheological disturbances (high shear stress) have an important role in promoting arterial thrombosis by enhancing the adhesive and signaling function of platelet integrin αIIbβ3 (GPIIb-IIIa). In this study we have defined a key role for the Type Ia phosphoinositide 3-kinase (PI3K) p110β isoform in regulating the formation and stability of integrin αIIbβ3 adhesion bonds, necessary for shear activation of platelets. Isoform-selective PI3K p110β inhibitors have been developed which prevent formation of stable integrin αIIbβ3 adhesion contacts, leading to defective platelet thrombus formation. In vivo, these inhibitors eliminate occlusive thrombus formation but do not prolong bleeding time. These studies define PI3K p110β as an important new target for antithrombotic therapy.

Cardiac Tissue Engineering in an In Vivo Vascularized Chamber

Background— Cardiac tissue engineering offers the prospect of a novel treatment for acquired or congenital heart defects. We have created vascularized pieces of beating cardiac muscle in the rat that are as thick as the adult rat right ventricle wall. Method and Results— Neonatal rat cardiomyocytes in Matrigel were implanted with an arteriovenous blood vessel loop into a 0.5-mL patented tissue-engineering chamber, located subcutaneously in the groin. Chambers were harvested 1, 4, and 10 weeks after insertion. At 4 and 10 weeks, all constructs that grew in the chambers contracted spontaneously. Immunostaining for &agr;-sarcomeric actin, troponin, and desmin showed that differentiated cardiomyocytes present in tissue at all time points formed a network of interconnected cells within a collagenous extracellular matrix. Constructs at 4 and 10 weeks were extensively vascularized. The maximum thickness of cardiac tissue generated was 1983 &mgr;m. Cardiomyocytes increased in size from 1 to 10 weeks and were positive for the proliferation markers Ki67 and PCNA. Connexin-43 stain indicated that gap junctions were present between cardiomyocytes at 4 and 10 weeks. Echocardiograms performed between 4 and 10 weeks showed that the tissue construct contracted spontaneously in vivo. In vitro organ bath experiments showed a typical cardiac muscle length-tension relationship, the ability to be paced from electrical field pulses up to 3 Hz, positive chronotropy to norepinephrine, and positive inotropy in response to calcium. Conclusion— In summary, the use of a vascularized tissue-engineering chamber allowed generation of a spontaneously beating 3-dimensional mass of cardiac tissue from neonatal rat cardiomyocytes. Further development of this vascularized model will increase the potential of cardiac tissue engineering to provide suitable replacement tissues for acquired and congenital defects.

Actions of intrathecal ω-conotoxins CVID, GVIA, MVIIA, and morphine in acute and neuropathic pain in the rat

Agents which decrease conductance of N-type voltage-gated Ca(2+) channels have been shown to attenuate measures of neuropathic pain in animal models and to provide symptom relief in humans. The omega-conotoxins have demonstrated efficacy but have a low therapeutic index. We have investigated the effects of a new omega-conotoxin, CVID (AM-336), and compared them with omega-conotoxin GVIA (SNX-124), omega-conotoxin MVIIA (SNX-111) and morphine in a spinal nerve ligation model of neuropathic pain in the rat. The ED(50) (and 95% CI) for attenuation of tactile allodynia by intrathecal administration for omega-conotoxin CVID, GVIA, MVIIA and morphine was 0.36 (0.27-0.48), 0.12 (0.06-0.24), 0.32 (0.23-0.45) and 4.4 (2.9-6.5) microg/kg, respectively. Only morphine significantly prolonged acute tail flick responses (ED(50) 2.3 (1.1-4.9) microg/kg). Of the omega-conotoxins, omega-conotoxin CVID showed the highest ratio of efficacy to behavioural toxicity. These observations show that intrathecal omega-conotoxins are effective in attenuating tactile allodynia in the rat without significantly affecting acute nociceptive responses. Omega-conotoxin CVID had similar potency to omega-conotoxin MVIIA but showed less toxicity in the therapeutic range.

Analysis of competitive agonist-antagonist interactions by nonlinear regression

The rigorous estimation of a dissociation constant (Kb) for antagonists in functional assays has been sought by pharmacologists using a variety of techniques ever since the regression method of Arunlakshana and Schild in 1959. Here, Michael Lew and James Angus describe a simplified global regression method with improved accuracy compared to Schild analysis. The method is suitable for personal computers with standard graphing and statistical software. The accuracy of the predicted pKb values and confidence intervals has been tested by comparing examples of published data, and by mathematical (bootstrap) simulations.

Pharmacology of Coronary Artery Bypass Grafts

Spasm of arterial and venous graft conduits can occur both during harvesting and after the graft is connected. Attempts to overcome spasm during harvesting by probing or hydraulic distension can cause structural damage to the graft, which may impair short- and long-term patency. After a coronary artery bypass graft is connected, spasm can cause major problems with myocardial perfusion. To select the best pharmacologic agent to prevent or reverse vasoconstriction in a graft requires an understanding of the reactivity of that particular type of graft to vasoconstrictor and vasodilator agents. The pharmacologic reactivity of venous and arterial graft conduits has been documented through extensive studies of isolated vessels in the organ bath and of in situ grafts in the body. In this review we summarize the current state of knowledge of the reactivity of arterial and venous grafts to vasoconstrictor and vasodilator agents and describe the practical application of this knowledge in the operating room and in the postoperative period.

Reactivity of the canine isolated internal mammary artery, saphenous vein, and coronary artery to constrictor and dilator substances : relevance to coronary bypass graft surgery

The internal mammary artery (IMA) and saphenous vein (SV) are used routinely in coronary artery (CA) bypass graft surgery. The IMA may develop spasm during surgery, and the SV often develops spasm during removal from the leg. We sought to determine the relative reactivity of the canine CA, IMA, and SV to potential vasoconstrictor substances and especially to determine which vasodilator agents were effective in these different blood vessels. All vessels were arranged as ring segments suspended at optimal stretch in organ baths. Glyceryl trinitrate (GTN) caused relaxation of the three vessels but was less sensitive, less potent (as determined by EC50 values), and had a reduced range of relaxation in the IMA. Papaverine was less sensitive in the IMA as compared with the CA and SV. Nifedipine, verapamil, and diltiazem were potent relaxing agents in all three vessels when precontracted by K+, but were less potent in vessels contracted by the thromboxane mimetic U46619 or phenylephrine, especially in the SV. These studies highlight the marked differences in the response of IMA and CA to constrictor and dilator agents and reinforce the notion that calcium antagonists of different chemical classes have widely differing activities in vascular tissue.

α2‐Adrenoceptors and endothelium‐dependent relaxation in canine large arteries

1 Ring preparations from the carotid, coronary, renal, mesenteric and femoral arteries of the dog were precontracted with the thromboxane mimetic U46619, after ensuring that the resting conditions were comparable from the Laplace relationship. 2 In the presence of prazosin (1 μm) and propranolol (3 μm), noradrenaline (NA) relaxed the arteries in the order coronary > carotid > femoral > renal = mesenteric. When maximum relaxation to nitroglycerin (10 μm) was taken to be 100% the maximum relaxation to noradrenaline in each artery was: coronary 70%; carotid 34%; femoral 19%; renal 7% and mesenteric 2%. 3 In endothelium‐intact arteries UK 14304 mimicked the relaxation responses to NA and idazoxan shifted the curves to both agonists to the right, consistent with an α2‐adrenoceptor classification. 4 Substance P relaxed the arteries in the same order as for NA but showed higher efficacy i.e.: coronary 100%; carotid 80%; femoral 71% renal 49%; and mesenteric 41%. Removal of the endothelium abolished the relaxation to NA. 5 We conclude that endothelium‐dependent relaxation to NA and substance P varies greatly across 5 large arteries of the dog. This may indicate that endothelium‐derived relaxing factor (EDRF) release is site‐dependent or that the efficacy of EDRF on smooth muscle varies; being greatest in the coronary and weakest in the renal and mesenteric arteries.

Evidence that CB-1 and CB-2 cannabinoid receptors mediate antinociception in neuropathic pain in the rat

&NA; The roles of the two cannabinoid receptor subtypes, CB‐1 and CB‐2, have not been clarified in cannabinoid‐mediated analgesia. We investigated the efficacy of the non‐selective cannabinoid receptor agonist CP55,940 in the modulation of responses in the rat to both acute pain (tail flick) and neuropathic pain (tactile allodynia following chronic L5/6 spinal nerve ligation). Responses were also assessed in the presence of the CB‐1 antagonist SR141716A (SR1) and the CB‐2 antagonist SR144528 (SR2). CP55,940 attenuated tactile allodynia (ED50 0.04 mg/kg i.t. (95% CI 0.032–0.044 mg/kg), 0.12 mg/kg i.p. (95% CI 0.10–0.15 mg/kg)) and induced thermal antinociception (ED50 tail flick 0.07 mg/kg i.t. (95% CI 0.05–0.10 mg/kg), 0.17 mg/kg i.p. (95% CI 0.11–0.26 mg/kg)). SR1 0.5 mg/kg i.t. attenuated the antinociceptive effect of CP55,940 in both modalities. However, SR1 1.0 mg/kg i.p. decreased tail flick latency but had no effect on tactile allodynia antinociception. In contrast, SR2 1.0 mg/kg i.p. significantly decreased the effect of i.p. CP55,940 on both tail flick antinociception and tactile allodynia (P<0.05). The combination of SR1 and SR2 (i.p.) had an additive effect in decreasing the antinociception induced by CP55,940 on tail flick responses (P<0.005). These results suggest a role for CB‐2 receptor‐mediated antinociception in both acute and neuropathic pain in addition to centrally located CB‐1 mechanisms.

Vasodilatation by acetylcholine is endothelium‐dependent: a study by sonomicrometry in canine femoral artery in vivo.

External diameter of the femoral artery was measured by sonomicrometry in the anaesthetized dog. Intra‐arterial acetylcholine lowered arterial pressure and thereby passively lowered diameter. When blood flow and distal resistance were controlled by roller pump and Starling resistor respectively, acetylcholine (0.1‐10 microM) and substance P (0.1‐1 nM) both caused up to 10% increase in diameter. Removal of endothelium by mechanically rubbing the artery lumen abolished the dilator response to acetylcholine and substance P but did not affect the response to nitroprusside. Constrictor responses to noradrenaline were unaltered by endothelium removal. Topical application of acetylcholine and substance P onto the adventitial surface of the artery also caused an increase in diameter but both agents were 50‐100 times less potent by this route compared with intra‐arterial infusion. These dilator responses were abolished by endothelium removal. In these circumstances acetylcholine caused constriction. We conclude that acetylcholine and substance P require an intact endothelium to elicit vasodilatation in vivo, at least for the large femoral artery. The results from the topical application experiments suggest that local neural release of vasoactive substances such as acetylcholine and substance P depend on an intact endothelium to cause vasodilatation.

STRUCTURE-FUNCTION RELATIONSHIPS OF OMEGA -CONOTOXIN GVIA : SYNTHESIS, STRUCTURE, CALCIUM CHANNEL BINDING, AND FUNCTIONAL ASSAY OF ALANINE-SUBSTITUTED ANALOGUES

The structure-function relationships of the N-type calcium channel blocker, ω-conotoxin GVIA (GVIA), have been elucidated by structural, binding and in vitro and in vivo functional studies of alanine-substituted analogues of the native molecule. Alanine was substituted at all non-bridging positions in the sequence. In most cases the structure of the analogues in aqueous solution was shown to be native-like by 1H NMR spectroscopy. Minor conformational changes observed in some cases were characterized by two-dimensional NMR. Replacement of Lys2and Tyr13 with Ala caused reductions in potency of more than 2 orders of magnitude in three functional assays (sympathetic nerve stimulation of rat isolated vas deferens, right atrium and mesenteric artery) and a rat brain membrane binding assay. Replacement of several other residues with Ala (particularly Arg17, Tyr22 and Lys24) resulted in significant reductions in potency (<100-fold) in the functional assays, but not the binding assay. The potencies of the analogues were strongly correlated between the different functional assays but not between the functional assays and the binding assay. Thus, the physiologically relevant assays employed in this study have shown that the high affinity of GVIA for the N-type calcium channel is the result of interactions between the channel binding site and the toxin at more sites than the previously identified Lys2 and Tyr13.