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Biomedical subjects

M R Dashwood

Publications and source records attributed to M R Dashwood.

At least 19 recordsLinked to original sources
PubMed2004

Down-regulation of nitric oxide synthase in the diabetic rabbit kidney: potential relevance to the early pathogenesis of diabetic nephropathy.

OBJECTIVES: Nephropathy is a well-recognised complication of diabetes mellitus (DM). The aim of this study was to investigate the effect of DM on the density and distribution of nitric oxide (NO) synthase (NOS) in the rabbit kidney. Quantification of the NOS radioligand on slide-mounted sections was compared with the nitroblue tetrazolium reaction, where the intensity of the reaction varies with the nicotinamide adenine dinucleotide diaphorase (NADPH-d) activity of NOS. MATERIALS AND METHODS: DM was induced with alloxan in six New Zealand White (NZW) rabbits. Plasma creatinine, urea and electrolytes were monitored at monthly intervals. The kidneys were removed following 6 months of DM. Transverse serial sections were cut and low-resolution autoradiography was performed using a radioligand for NOS ([(3)H]-NOARG). Histochemical localisation of NADPH-d activity was also performed. Densitometric analysis was performed on the autoradiographs and the results compared with those obtained from six age-matched control rabbits. RESULTS: There was a significant (p < 0.01) rise in plasma creatinine levels in the diabetic rabbits, although the mean values remained within the reference range. There was a significant (p < 0.0001) down-regulation of NOS binding sites in both the cortex and medulla of the DM kidney when compared with the controls. A similar decrease in NADPH-d activity was seen in the diabetic renal cortex and medulla. In addition, NADPH-d activity also appeared to be reduced in the diabetic glomeruli when compared with controls. CONCLUSIONS: NOS binding sites and NADPH-d activity are significantly decreased in the DM renal cortex and medulla. These changes are associated with a mild deterioration in renal function and may be an early event that could subsequently play a role in the progression of DM nephropathy. Manipulating the NO pathway during the early stages of DM nephropathy may be beneficial.

Animals↗
PubMed2004

Human saphenous vein and coronary bypass surgery: ultrastructural aspects of conventional and "no-touch" vein graft preparations.

Coronary artery bypass graft surgery (CABG) is routinely used to restore blood flow to diseased cardiac muscle due to coronary artery disease. The patency of conventional grafts decreases with time, which is due to thrombosis and formation of neointima. A primary cause of graft failure is the mechanical damage inflicted to the graft during harvesting, including removal of surrounding tissue accompanied by high pressure saline distension to overcome vasospasm (both causing considerable mechanical trauma). The aim of this study was to compare the ultrastructural features of human saphenous vein (SV) grafts harvested conventionally and grafts prepared using an atraumatic 'no-touch' harvesting technique introduced by Souza (1996). The results of this study showed a better preservation of the lumenal endothelium and medial vascular smooth muscle (SM) in 'no-touch' versus conventional grafts. A 'fast' (within 30 min) response of SM cells to conventional harvesting was noted where features of both SM cell division and apoptosis were observed. It is concluded that the 'preserved' nature of the 'no-touch' aortocoronary SV grafts renders them less susceptible to thrombotic and atherosclerotic factors than grafts harvested conventionally. These features are suggested to contribute to the improved early patency rate described using the no-touch technique of SV harvesting.

Aged↗
PubMed2003

Detection of glucagon receptor mRNA in the rat proximal tubule: potential role for glucagon in the control of renal glucose transport.

Glucagon is known to affect glomerular filtration rate and renal tubular solute and fluid transport, although it is only thought to act directly on the thick ascending limb (TAL) and collecting duct (CD). Indeed, previous studies have detected glucagon-sensitive adenylate cyclase exclusively in these nephron segments, suggesting the presence of glucagon receptors. In the present study, we have demonstrated for the first time that glucagon receptor mRNA is expressed in the rat proximal tubule, as well as in the TAL and CD. By autoradiography, we have also shown that specific binding of glucagon occurs in both the renal cortex and medulla. In addition, using proximal tubule brush-border membrane (BBM) vesicles for studies of glucose transport, we have established that glucagon stimulates glucose uptake via a facilitative GLUT-mediated transport process (by 58%; P < 0.005), whereas cAMP stimulates only the sodium glucose-linked transporter ('SGLT')-mediated glucose uptake (by 53%; P < 0.05). Taken together, these findings suggest that glucagon could have a role in controlling proximal tubular transport function, including glucose reabsorption, but unlike in the TAL and CD, the proximal tubule glucagon receptor might not be coupled primarily to adenylate cyclase.

Animals↗
PubMed2003

Evidence for the involvement of endothelin-1 but not urotensin-II in chronic lower limb ischaemia in man.

BACKGROUND: endogenous vasoconstrictor peptides may play a role in the pathophysiology of critical limb ischaemia (CLI). This study investigated endothelin-1 (ET-1) and urotensin-II (U-II) mRNA expression, peptide distribution and ET receptor subtype binding in chronically ischaemic muscle. METHODS: open muscle biopsies were taken from patients undergoing amputations for CLI and from patients undergoing coronary artery bypass surgery (controls). ET-1 and U-II mRNA expression in muscle biopsies was studied using real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). ET-1 and U-II immunohistochemistry was performed on muscle sections and ET receptor binding studied using in vitro autoradiography. RESULTS: ET-1 mRNA expression was significantly increased in CLI compared to controls (p<0.05) whilst no significant change in U-II expression occurred. ET-1 immunoreactivity was also increased in CLI with no difference in U-II immunostaining observed. ET(B) receptor binding was significantly increased in CLI (median 4, range 1-8 vs 2, range 1-3, dpm x 10(3)/mm(2), p=0.01, Mann-Whitney test) whilst ET(A) receptor binding was not significantly raised. Binding was associated with microvessels and macrophages. CONCLUSIONS: in CLI, the ET-1 pathway is upregulated but U-II is unaffected. ET-1 may vasoconstrict microvessels and mediate inflammation in chronically ischaemic muscle. ET-1 binding to ET(B) receptors in particular may play an important role in the pathophysiology of CLI underscoring the therapeutic potential of ET(B) receptor antagonists in the management of CLI.

Aged↗
PubMed2003

Endothelin-1 and nitric oxide in the pathogenesis of urinary tract disorders secondary to bladder outlet obstruction.

Bladder outlet obstruction (BOO) is a common disorder that is associated with urinary tract symptoms. Nitric oxide (NO), synthesized by NO synthase (NOS) is a potent vasodilator that is present throughout the urinary tract and the corpus cavernosum. Endothelin-1 (ET-1) conversely is a potent vasoconstrictor peptide that is similarly distributed throughout the urinary tract. ET-1 and NO as well as possessing opposing actions regulate each other's synthesis. The disruption of the balance between ET-1 and NO is associated with various vascular pathologies. However, their potential roles in the pathogenesis of urinary tract disorders, secondary to BOO, is not well established. New Zealand White rabbits with BOO are considered to be a suitable model of the human condition. Hence, using this model, we systematically investigated the potential roles of ET-1 and NO in the pathogenesis of the various urological disorders associated with BOO. In this review we discuss the results of our studies, which support the concept that an imbalance between ET-1 and NO may be associated with the pathogenesis of urinary tract disorders secondary to BOO. We also discuss the potential clinical implications of this association. This review is based on the Bard Silver Medal Lecture given (by MAK) at the 2002 British Association of Urological Surgeons (BAUS) annual meeting.

Animals↗
PubMed2002

Enhanced relaxation of diabetic rabbit cavernosal smooth muscle in response to nitric oxide: potential relevance to erectile dysfunction.

New Zealand white rabbit cavernosal smooth muscle strips (n=6) were mounted in organ baths. Relaxations to nitric oxide (10(-7)-10(-4) mol/l) were measured and the same procedure was repeated on strips from rabbits 6 months after alloxan-induced diabetes (n=6). Transverse cavernosal sections were obtained from the same penises. Low and high resolution autoradiographs were prepared using [(3)H]-L-N(G)-nitroarginine (an index of nitric oxide binding sites) and analysed densitometrically. Histochemical analysis was performed on adjacent sections using NADPH diaphorase (an index of nitric oxide synthase activity). Nitric oxide relaxed control rabbit cavernosal smooth muscle strips in a concentration-dependent manner. Diabetic rabbit cavernosal smooth muscle strips were significantly (P<0.03) more sensitive to nitric oxide (mean IC(50)=3.9 x 10(-6) mol/l). Nitric oxide synthase binding sites were localised to the cavernosal endothelium and smooth muscle. Nitric oxide synthase activity was increased in 6 month diabetic cavernosal smooth muscle. These findings suggest impairments in the L-arginine-nitric oxide pathway may play a role in the pathophysiology of diabetic erectile dysfunction.

Acetylcholine↗
PubMed2002

Potential role of endothelin 1 in ischaemia-induced angiogenesis in critical leg ischaemia.

BACKGROUND: Ischaemia-induced angiogenesis occurs in critical leg ischaemia (CLI) and endothelin (ET) 1 may be involved in this process. The aim of this study was to quantify microvessels and study ET receptor expression and distribution in critically ischaemic leg muscle. METHODS: Leg muscle biopsies were taken from 12 patients with CLI and 12 patients with no leg ischaemia. Microvessels were identified immunohistochemically on muscle sections, and the number of immunopositive cells was quantified. ETA and ETB receptor messenger RNA (mRNA) expression was studied using real-time quantitative reverse transcriptase-polymerase chain reaction, and receptor binding was localized and assessed by in vitro autoradiography. RESULTS: The number of microvessels in CLI muscle biopsies was 2.6 times higher than that in controls (P < 0.01). ETB receptor mRNA expression and binding were significantly increased in CLI tissue (P < 0.05), while ETA receptor levels were not significantly raised. High-resolution autoradiography showed that ET receptor binding was associated with microvessels. CONCLUSION: Angiogenesis occurs in CLI and raised ETB receptors within the muscle were associated with microvessels, suggesting that ET-1 may mediate angiogenesis via these receptors in critically ischaemic muscle.

Aged↗
PubMed2002

Endothelin receptors in the aetiology and pathophysiology of varicose veins.

INTRODUCTION: varicose veins are tortuous and poorly contractile. Their aetiology remains unclear. Neovascularisation has been suggested as a possible explanation. Endothelins are mitogenic, promoting proliferation and migration of endothelial cells via endothelin-B receptors. We hypothesise that endothelial cells and endothelin receptor density and distribution may play a role in the development of varicosis. METHODS: saphenous vein segments from nine patients with varicose veins were compared to six controls. Slide-mounted sections were incubated in radioactive labelled endothelin-1 and receptor subtype-selective ligands and binding sites assessed using autoradiography. Endothelin-1 and endothelial cells were identified by immunohistochemistry and CD31-positive staining cells counted. RESULTS: radioactive labelled endothelin-1 and endothelin-B receptor binding was reduced in varicose compared to control veins (p=0.04). Endothelin-A receptor binding was diffuse, with no difference in density in both groups (p=0.58). Endothelin-B receptor binding was diffuse with superimposed clusters. Although the density of medial endothelin-B receptor binding was reduced in the varicose group, more clusters were identified in this group compared to controls (p=0.005). CD-31 staining identified these clusters as endothelial cells. CONCLUSION: the reduced endothelin-1 binding and endothelin-B receptor density may be partially responsible for the reduced vasocontractility in varicose veins. We speculate that the increase in endothelin-B receptor binding CD31-positive endothelial cells in varicose veins may potentially stimulate mitogenesis and migration, leading to new vessel formation.

Binding, Competitive↗
PubMed2002

Recent strategies to reduce vein graft occlusion: a need to limit the effect of vascular damage.

Despite early identification and aggressive modification of atherosclerotic risk factors, many patients still require surgical revascularisation for established atherosclerotic vascular disease. However, bypass surgery is hampered by a high incidence of vein graft failure. New strategies are being introduced to improve these results, with early data suggesting that improved patency rates are possible. These vary from the use of adjuvant pharmacological agents and local gene transfer strategies to the modification of vein harvesting techniques in order to reduce vascular damage to all layers of the graft. Advances in vascular biology have resulted in new insights into the role of the endothelium and adventitia in vein graft remodelling. Although recent pharmacological adjuvant therapy and molecular techniques have been described that may be used to reduce the incidence of vein graft occlusion a more desirable approach for improved graft patency rates may be achieved simply by using atraumatic surgical techniques aimed at minimising vascular damage during vessel harvesting and subsequent anastamoses during bypass surgery.

Animals↗
PubMed2001

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.

Adult↗
PubMed2001

Preserved endothelial integrity and nitric oxide synthase in saphenous vein grafts harvested by a 'no-touch' technique.

BACKGROUND: The saphenous vein is the most commonly used conduit for coronary artery bypass surgery, but 1-year occlusion rates as high as 30 per cent have been reported. In conventional surgery, considerable damage to the vein occurs during harvesting. The aim of this study was to compare endothelial integrity and nitric oxide synthase (NOS) in saphenous veins harvested by a novel 'no-touch' technique and veins harvested conventionally. METHODS: Immunohistochemistry was used to study endothelial integrity, and a combination of histochemistry and autoradiography was employed to identify NOS in human saphenous veins harvested by conventional and no-touch techniques. RESULTS: The endothelial lining of conventional grafts was reduced compared with that of no-touch grafts (52 versus 73 per cent; P = 0.04). This was associated with a concomitant reduction of NOS availability; NOS was also present in the adventitial vasa vasorum of no-touch vessels. CONCLUSION: Some of the sites with potential for nitric oxide release in vivo are removed during conventional saphenous vein harvesting. These sites were preserved after no-touch harvesting, suggesting the potential to improve coronary artery bypass graft patency.

Aged↗
PubMed2001

Fibroblast matrix gene expression and connective tissue remodeling: role of endothelin-1.

This study examines endothelin-induced modulation of extracellular matrix synthesis and remodeling by fibroblasts, and its potential role in the pathogenesis of systemic sclerosis (scleroderma). Endothelin-1 promoted fibroblast synthesis of collagen types I and III, but not fibronectin, by a mechanism dependent upon both ETA and ETB receptors. Conversely, endothelin-1 inhibited both protein expression of matrix metalloproteinase 1 and zymographic activity exclusively via ETA receptors. A dual regulatory role for endothelin-1 in transcriptional regulation was suggested by the ability of endothelin-1 to enhance steady-state levels of collagen mRNA and activate the proalpha2(I) collagen (Col1a2) promoter, but in contrast to reduce matrix metalloproteinase 1 transcript expression and suppress transcription of a human matrix metalloproteinase 1 promoter reporter construct in transient transfection assays. Although endothelin-1 significantly enhanced remodeling of three-dimensional collagen lattices populated by normal fibroblasts, this was not observed for lattices populated by systemic sclerosis fibroblasts. Promotion of matrix remodeling was dependent upon ETA receptor expression and was blocked by specific inhibitors of tyrosine kinases or protein kinase C. Reverse transcriptase polymerase chain reaction, S1 nuclease, and functional cell surface binding studies showed that normal and systemic sclerosis fibroblasts express both ETA and ETB receptors (predominantly ETA), but that ETA receptor mRNA levels and ETA binding sites on fibroblasts cultured from systemic sclerosis skin biopsies are reduced by almost 50%. Endothelin-1 is thus able to induce a fibrogenic phenotype in normal fibroblasts that is similar to that of lesional systemic sclerosis fibroblasts. Moreover, reduced responsiveness to exogenous endothelin-1 in systemic sclerosis suggests that downstream pathways may have already been activated in vivo. These data further implicate dysregulated endothelin-receptor pathways in fibroblasts in the pathogenesis of connective tissue fibrosis.

Cells, Cultured↗
PubMed2000

Doxazosin modifies serotonin-mediated rabbit urinary bladder contraction. Potential clinical relevance.

5-Hydroxytryptamine (5-HT) induces rabbit detrusor contractions via 5-HT3 receptors. Similarly, 5-HT4 receptors are known to be present in the human bladder. Doxazosin, a non-selective alpha1 antagonist, is used for the symptomatic relief of bladder outflow obstruction. Previous work has shown that doxazosin inhibits 5-HT2-mediated platelet shape change. Hence, the aim of this study was to assess, using organ baths and autoradiography, whether doxazosin has any 5-HT-inhibiting activity in the rabbit detrusor. Detrusor strips from adult New Zealand White rabbits were placed in organ baths; phenoxybenzamine (10(-5) M) was added to block alpha-receptors. After KCl responses were assessed, the tissues were exposed to 10(-3) M 5-HT. Subsequently, the strips were incubated with doxazosin or ondansetron (10(-5) M; 5-HT3 antagonist) followed by a further exposure to 5-HT. In some experiments, after the initial 5-HT-induced contractions, the tissues were washed and then re-exposed to 5-HT. These latter experiments acted as controls. Low-resolution autoradiography was performed on detrusor sections to assess the effect of doxazosin on 5-HT binding. These sections were analyzed densitometrically. Doxazosin and ondansetron produced a significant reduction in 5-HT-mediated contractions. Inhibition by doxazosin was in a concentration-dependent manner. Autoradiography demonstrated a significant reduction in [3H]-5-HT binding by doxazosin. Doxazosin significantly inhibits 5-HT-mediated contractions in the rabbit detrusor. This effect appears to be mainly mediated via 5-HT3 receptor inhibition. Autoradiographic evidence suggests that doxazosin reduces 5-HT binding in the rabbit detrusor. The beneficial effects of doxazosin in bladder outflow obstruction may be due, at least in part, to 5-HT antagonism.

Animals↗
PubMed2000

Structural and cellular adaptation of duodenal iron uptake in rats maintained on an iron-deficient diet.

Iron deficiency induced in rats maintained on a commercial diet with a low iron content has been used to investigate adaptive mechanisms that enhance duodenal iron uptake. These adaptive changes have been divided into those that result from changes in villus surface area (structural adaptation) and those that reflect changes in the way individual enterocytes express iron transport function (cellular adaptation). Cellular adaptation was assessed by carrying out microdensitometry of autoradiographs prepared from duodenal tissue previously incubated for 5 min in 200 micromol/l 59Fe2+-ascorbate. Structural adaptation was studied by performing image analysis of microdissected and sectioned villi. Cellular adaptation involved increased iron uptake by enterocytes present in the lower villus. Thus iron deficiency resulted in a threefold enhanced expression of uptake in the lower 100 microm villus (3.9+/-2.4 versus 12.6+/-1.5 arbitrary units, P<0.001). Maximal uptake was reached in the upper region of both control and iron-deficient villi, but iron deficiency had no effect on cellular uptake at this part of the villus. Structural adaptation involved the lengthening (+16%, P<0.05) and broadening (+14%) of villi in the duodenum of iron-deficient rats. The resultant expansion in villus area caused a further increase in uptake that was mostly expressed in the upper villus. Maximal uptake corrected for structure occurred in the middle third of villi from control and iron-deficient rats. Cellular plus structural adaptation produced a twofold increase in iron uptake. More than half of this effect was caused by changes in villus structure. [3H]Thymidine labelling experiments revealed a slightly earlier expression of enterocyte iron uptake in iron deficiency.

Adaptation, Physiological↗
PubMed2000

Neural reorganisation in porcine vein grafts: a potential role for endothelin-1.

Vein grafts are associated with adventitial remodelling which may influence innervation of the graft. Since there is also evidence that endothelin-1 (ET-1) plays a role in the adventitial remodelling process, we investigated neural distribution in porcine vein grafts 1 and 6 months after implantation, as well as the localisation of immunoreactive ET-1 and its receptors in the same tissues. Saphenous vein-carotid artery interposition grafting was performed in Landrace pigs. One and 6 months after surgery, vein grafts and ungrafted saphenous veins were excised; neural tissue and ET-1 were identified by immunocytochemistry and ET receptors were identified using in vitro autoradiography. In ungrafted saphenous veins, abundant perivascular nerves were located in the outer region of the media with only a few paravascular nerves in the adventitia. In vein grafts at 1 month after implantation, there was almost complete depletion of perivascular nerves in the media. In contrast, in the neoadventitia, there was an emphatic appearance of large paravascular nerve bundles and a marked increase in small paravascular nerves. These changes were more pronounced at 6 months after surgery, although the principal changes had occurred within 1 month. Immunoreactive ET-1 (index of ET-1 content) was associated with paravascular nerve bundles, appearing as a dark, dense ring at the perineurium. Furthermore, within the nerve bundles, positive ET-1 immunoreactivity was associated with positive alpha-actin staining, indicating that ET-1 is associated with (neural) microvessels. Also, dense 125I-labelled BQ3020 (ET(B)-selective) binding to nerve bundles was observed, indicating the presence of ET(B) receptor subtypes. ET(A) receptor subtypes were not detected in neural tissue. These data demonstrate neural reorganisation in vein grafts and indicate that ET-1 content and binding may play a role in this process. The functional consequences of these changes on neointima formation, a major cause of vein graft failure, remain to be determined.

Animals↗
PubMed2000

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.

Adenosine↗