Michael R. Dashwood

α2-Adrenoceptor modulation of nociception in rat spinal cord: location, effects and interactions with morphine

The effects of intrathecal clonidine alone and prior to intrathecal morphine were studied on electrically evoked A beta and C fibre activity in the dorsal horn of the halothane-anaesthetised rat. Clonidine reduced C fibre-evoked activity in a dose-dependent manner, to a maximum 52% inhibition which was reversed by rauwolscine and yohimbine but not naloxone. High doses of clonidine also produced small inhibitions of A fibre-evoked activity. Clonidine potentiated the inhibitory action of intrathecal morphine on electrically evoked C fibre activity but not A fibre activity. In addition, the location of alpha 2-adrenoceptor and opiate binding sites in consecutive sections of rat lumbar cord was investigated using in vitro autoradiography with selective ligands, and it was demonstrated that both opiate and alpha 2-receptor types are present within the same superficial layers of the dorsal horn of the same animal. The results indicate that alpha 2-adrenoceptors and opiate receptors can interact in the modulation of nociceptive transmission in rat spinal cord.

Constitutive ALK5-Independent c-Jun N-Terminal Kinase Activation Contributes to Endothelin-1 Overexpression in Pulmonary Fibrosis: Evidence of an Autocrine Endothelin Loop Operating through the Endothelin A and B Receptors

ABSTRACT The signal transduction mechanisms generating pathological fibrosis are almost wholly unknown. Endothelin-1 (ET-1), which is up-regulated during tissue repair and fibrosis, induces lung fibroblasts to produce and contract extracellular matrix. Lung fibroblasts isolated from scleroderma patients with chronic pulmonary fibrosis produce elevated levels of ET-1, which contribute to the persistent fibrotic phenotype of these cells. Transforming growth factor β (TGF-β) induces fibroblasts to produce and contract matrix. In this report, we show that TGF-β induces ET-1 in normal and fibrotic lung fibroblasts in a Smad-independent ALK5/c-Jun N-terminal kinase (JNK)/Ap-1-dependent fashion. ET-1 induces JNK through TAK1. Fibrotic lung fibroblasts display constitutive JNK activation, which was reduced by the dual ETA/ETB receptor inhibitor, bosentan, providing evidence of an autocrine endothelin loop. Thus, ET-1 and TGF-β are likely to cooperate in the pathogenesis of pulmonary fibrosis. As elevated JNK activation in fibrotic lung fibroblasts contributes to the persistence of the myofibroblast phenotype in pulmonary fibrosis by promoting an autocrine ET-1 loop, targeting the ETA and ETB receptors or constitutive JNK activation by fibrotic lung fibroblasts is likely to be of benefit in combating chronic pulmonary fibrosis.

Differential Leukotriene Constrictor Responses in Human Atherosclerotic Coronary Arteries

BACKGROUND Leukotrienes are a class of biologically active lipids that have potent effects on the heart. To assess their role in coronary artery disease, we compared the contractile responses of leukotriene C4 (LTC4) and leukotriene D4 (LTD4) and their binding activity in both atherosclerotic and nonatherosclerotic human coronary arteries. We also studied expression of the enzymes that control their formation to understand how the 5-lipoxygenase (5-LO) pathway is activated in the coronary arteries. METHODS AND RESULTS The capacity of leukotrienes to affect coronary vessel tone and the influence of atherosclerosis was tested in organ baths. Leukotriene receptors were examined by autoradiography, and antibody binding to the various enzymes responsible for their formation was assessed by use of immunocytochemistry. Nonatherosclerotic coronary artery ring segments were unresponsive to LTC4 and LTD4. In contrast, LTC4 and LTD4 induced concentration-dependent contractions in atherosclerotic coronary arteries. Specific [3H]-LTC4 but not LTD4 binding to atherosclerotic coronary artery was evident, with no evidence of specific binding of [3H]-leukotrienes to nonatherosclerotic coronary artery. High-resolution autoradiography identified specific [3H]-LTC4 binding sites to smooth muscle cell and to regions of intimal proliferation and plaque. Cells showing positive antibody binding to 5-LO, FLAP (5-lipoxygenase activating protein), and leukotriene A4 hydrolase were also present in the coronary arteries and had a similar distribution to macrophages. CONCLUSIONS Atherosclerosis is associated with a specific leukotriene receptor(s) capable of inducing hyperreactivity of human epicardial coronary arteries in response to LTC4 and LTD4.

Differential cardiovascular and respiratory responses to central administration of selective opioid agonists in conscious rabbits: correlation with receptor distribution.

1 The effects of intracerebroventricular (i.c.v.) and intracisternal (i.c.) administration of a range of doses (0.01, 0.1 and 1.0 nmol kg−1) of specific μ‐ δ‐ and κ‐opioid agonists on cardiovascular and respiratory function and on plasma catecholamines have been studied in conscious rabbits. The distribution of μ‐ δ‐ and κ‐opioid receptors was localized in rabbit brain by in vitro autoradiography. 2 The μ‐agonist [D‐Ala2, MePhe4‐Gly5‐ol]enkephalin (DAGOL) given i.c.v. caused a large rise in plasma noradrenaline and adrenaline, hypertension accompanied by an initial bradycardia followed by tachycardia, respiratory depression and sedation. After i.c. administration there were similar changes in heart rate (HR) and respiration, but no significant changes in mean arterial pressure (MAP) or plasma catecholamines. 3 The δ‐agonist [D‐Pen2,5]enkephalin (DPDPE) increased MAP and HR after both i.c.v. and i.c. administration, caused a small increase in noradrenaline but had no effect on adrenaline and did not alter respiration rate or blood gases. After i.c.v. DPDPE the rabbits became more alert and active. 4 The κ‐agonist U69593 given i.c.v. or i.c. had no effect on MAP or HR. After i.c.v. U69593, Paco2 fell, but there were no other respiratory effects. The responses to dynorphin 1–13, an endogenous κ‐agonist, were similar to those of U69593. 5 The opioid antagonist naloxone (30 nmol kg−1) given intravenously (i.v.) blocked the effects of i.c.v. DAGOL (1 nmol kg−1). A 100 fold higher dose of i.v. naloxone (3 μmol kg−1) was required to abolish the effects of i.c.v. DPDPE (1 nmol kg−1). 6 Autoradiographic studies demonstrated a high density of μ‐ and δ‐opioid receptors in hypothalamic sites. In the brainstem μ‐receptors were demonstrated in the nucleus tractus solitarius (NTS) and δ‐receptors in the dorsal motor nucleus of the vagus. κ‐Receptors were not detected in either the hypothalamus or brainstem. 7 These findings demonstrate that DAGOL increases sympatho‐adrenal outflow, probably by stimulation of hypothalamic μ‐receptors. The effects on HR are probably partly through a baroreflex and partly through an action of DAGOL on μ‐receptors in the dorsal motor nucleus of the vagus. DPDPE probably acts on δ‐receptors in the NTS to increase MAP and HR. Respiratory depression resulted from stimulation of μ‐receptors in the brainstem with no evidence of δ‐ or κ‐receptors being involved.

Autoradiographic localisation of opiate receptors in rat small intestine

[3H]Naloxone and [3H]dihydromorphine are selective ligands for opiate receptors. Using an in vitro autoradiographic technique, binding of these ligands has been demonstrated to the villi and crypts in rat small intestine. These results indicate the presence of opiate receptor sites in the small intestine which suggests further a role for endogenous opiates in the transport functions of intestinal mucosa.

High early patency of saphenous vein graft for coronary artery bypass harvested with surrounding tissue

BACKGROUND Surgical trauma to the saphenous vein, used as a conduit for coronary artery bypass grafting, affects their occlusion rate. This study evaluates the early patency of saphenous vein grafts harvested with a pedicle of surrounding tissue that protects the vein from spasm and trauma. METHODS Fifty-two patients underwent coronary artery bypass grafting with saphenous veins harvested with surrounding tissue. Forty-five patients, who received a total of 124 vein grafts and 42 left internal mammary arteries, underwent angiographic follow-up at a mean of 18 months (9 to 24 months). RESULTS Patency for saphenous vein grafts was 95.4% and for left internal mammary arteries, it was 93.3%. Twenty-nine of 30 (96.7%) vein grafts anastomosed to arteries 2.0 mm or more, 65 of 67 (97%) grafts to 1.5 mm, and 10 of 13 (77%) anastomosed to 1-mm arteries were patent. Nineteen of 22 (86.4%) vein grafts with flow rates 20 mL/min or less, 32 of 34 (94.1%) with flow between 20 and 40 mL/min, and 50 of 51 (98%) with flow more than 40 mL/min were patent. Other registered surgical and clinical factors did not contribute to vessel occlusion. CONCLUSIONS Early patency rate of saphenous veins harvested with surrounding tissue is very high, even in saphenous vein grafts demonstrating low blood flow. Preservation of graft endothelium using our harvesting technique may be the explanation of this success.

Does Periadventitial Fat-Derived Nitric Oxide Play a Role in Improved Saphenous Vein Graft Patency in Patients Undergoing Coronary Artery Bypass Surgery?

Background/Aims: The saphenous vein is commonly used for coronary artery bypass surgery but its patency is poor. Vascular damage occurs during conventional surgery. However, patency improves when the graft is harvested with minimal surgical trauma, partly due to preservation of vascular endothelial nitric oxide synthase (eNOS) and tissue sources of nitric oxide (NO), a factor possessing both dilatory and anti-proliferative properties. Apart from these grafts exhibiting an intact luminal endothelium they are harvested complete with a surrounding cushion of tissue, much of which is fat. Methods: Immunostaining for eNOS was performed on vein graft sections and reverse-transcriptase polymerase chain reaction and Western blotting were used to identify eNOS mRNA and protein. NO synthase activity was measured using the citrulline assay. Results: Immunohistochemistry identified eNOS staining of vein graft segments, including dense staining of the cushion of perivascular fat and associated structures surrounding the vein. eNOS protein was confirmed in both the vein and surrounding fat by Western blot analysis. Using the citrulline assay, the perivascular fat and underlying vein possessed comparable NO synthase activity. Conclusions: Our observations suggest that perivascular fat-derived NO plays a beneficial role in saphenous veins harvested atraumatically and used as grafts in patients undergoing coronary artery bypass surgery.

Localization and action of adenosine A2a receptors in regions of the brainstem important in cardiovascular control

In vitro autoradiography and central microinjections of a P1 adenosine A2a receptor antagonist have been employed to investigate a possible role for centrally located adenosine A2a receptors in modulation of the baroreceptor reflex. In vitro autoradiography using [125I]4-(2-[7-amino-2-[2-furyl][3,2,4]triazolol[2,3-a][1,3,5]tr iazin-5-yl-amino]ethyl)phenol ([125I]ZM241385), the high-affinity adenosine A2a receptor antagonist, revealed a heterogeneous distribution of adenosine A2a binding sites within the lower brainstem of the rat. Image analysis showed high levels of binding in rostral regions of both the nucleus tractus solitarius and the ventrolateral medulla. Intermediate levels of binding were observed in the commissural nucleus tractus solitarius and the dorsal vagal motor nucleus, with low levels of binding in caudal regions of the nucleus tractus solitarius and the ventrolateral medulla, and the hypoglossal nucleus. Unilateral microinjections of unlabelled ZM241385 into the nucleus tractus solitarius had no effect on baseline levels of arterial pressure, heart rate and phrenic nerve activity recorded in anaesthetized, artificially ventilated rats. However, microinjections of ZM241385 reduced the bradycardia evoked by stimulation of the ipsilateral aortic nerve. In contrast, ZM241385 had no effect on the depressor response or the reduction in phrenic nerve activity evoked by aortic nerve stimulation. Our results indicate that adenosine A2a binding sites are located in a number of brainstem regions involved in autonomic function, consistent with the idea that adenosine acts as a neuromodulator of a variety of cardiorespiratory reflexes. Specifically, the data support the hypothesis that adenosine A2a receptors located within the nucleus tractus solitarius are activated during baroreceptor stimulation and have an important modulatory role in the pattern of cardiovascular changes associated with this reflex.

[125I]-endothelin-1 binding to vasa vasorum and regions of neovascularization in human and porcine blood vessels: a possible role for endothelin in intimal hyperplasia and atherosclerosis.

The distribution of [125I]-endothelin-1 (ET-1) binding sites on human and porcine vessels was studied with in vitro receptor autoradiography. Binding to normal human saphenous veins was compared to atheromatous veins used as coronary artery bypass grafts. Binding to porcine vessels, from an experimental model of intimal hyperplasia, was also studied. There was dense binding of [125I]-ET-1 to smooth muscle of all vessels examined, as well as to the vasa vasorum and regions of neovascularization of diseased vessels. Binding to microvasculature (vasa vasorum and regions of neovascularization) is of particular interest, because ET-1 has been shown to have mitogenic activity on vascular smooth-muscle cells in culture and microvessels are extremely sensitive to the constrictor effect of ET-1. Binding of [125I]-ET-1 to vasa vasorum of normal blood vessels and to regions of neovascularization of atheromatous vessels suggests that ET-1 plays a pathophysiologic role in atherosclerosis.

Distribution of endothelin-1 (ET) receptors (ETA and ETB) and immunoreactive ET-1 in porcine saphenous vein–carotid artery interposition grafts

Proliferation of vascular smooth muscle cells (VSMC) is a principal event in neointima formation in saphenous vein-coronary artery bypass grafts. Since endothelin-1 (ET-1) promotes VSMC replication and ET-1 receptor antagonists inhibit neointima formation in arterial injury models, it is reasonable to propose that ET-1 may be involved in neointima formation in vein grafts. However, it is not known what alterations of ET-1 and its receptors (if any) occur in vein grafts. The objective of this study, therefore, was to investigate the distribution of ET-1 and ET-1 receptor subtypes (ET(A) and ET(B)) in porcine vein grafts. Unilateral interposition saphenous vein grafting was performed by end to end anastomosis after excision of a segment of carotid artery in Landrace pigs. One month after surgery, vein grafts, ungrafted saphenous veins and carotid arteries were excised, ET-1 immunoreactivity identified by immunocytochemistry and ET(A) and ET(B) receptor subtypes studied using autoradiography. In vein grafts, there was a greater density of ET(A) compared to ET(B) receptors in both the tunica media and neointima. ET(A) binding in the tunica media of ungrafted saphenous vein was greater than that in the carotid artery or vein grafts, but greater in the vein graft compared to the carotid artery. Immunoreactive ET-1 was located in endothelial cells and throughout the neointima of the vein graft. Dense ET-1 binding (to both ET(A) and ET(B) receptors) was also associated with microvessels in the adventitia within the graft. In vein grafts, there was strong ET(B) binding to neutrophils which were present in high numbers at the subendothelium and within the adventitia. It is concluded ET(A) receptors may play a role in vein graft thickening at the medial and neointimal VSMC level, whereas ET(B) receptors may play a role in microangiogenesis. The higher levels of ET(A) receptors in the tunica media of ungrafted saphenous vein relative to the carotid artery and vein graft may also render this conduit susceptible to neointima formation. These data indicate that studies on the effect of ET receptor antagonists on the pathobiology of vein graft disease is warranted.