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Biomedical subjects

D W Gray

Publications and source records attributed to D W Gray.

At least 91 records · Page 5Linked to original sources
PubMed1992

Nitric oxide synthesis inhibitors attenuate calcitonin gene-related peptide endothelium-dependent vasorelaxation in rat aorta.

The effect of inhibitors of nitric oxide synthesis were examined on the endothelium-dependent relaxation induced by acetylcholine and human alpha-calcitonin gene-related peptide (CGRP) on rat isolated aortic rings preconstricted with noradrenaline. The endothelium-dependent vasorelaxation induced by acetylcholine and CGRP was inhibited by NG-monomethyl-L-arginine (L-NMMA) and NG-nitro-L-arginine (L-NOARG) in a concentration-related fashion. The inhibition of endothelium-dependent relaxation by L-NMMA and L-NOARG was partially reversed by L-arginine but not by D-arginine for both acetylcholine and CGRP. The data presented suggests that CGRP, like acetylcholine; relaxes the rat aorta via the release of nitric oxide.

Acetylcholine↗
PubMed1992

A prospective, randomized, blind comparison of three biopsy techniques in the management of patients after renal transplantation.

Although conventional histology (CH) of needle core biopsies has been accepted as the gold standard for diagnosis of renal allograft rejection, this assumption has never been tested. Fine-needle aspiration cytology (FNAC) and monoclonal antibody (panleukocyte) staining of needle core biopsies (MABS) have been suggested to be superior to CH. A total of 50 patients received a cadaveric renal transplant followed by immunosuppression with triple therapy. Biopsies were taken routinely at days 7,14,21,28, and 90, with additional biopsies taken between these times if rejection was suspected (total biopsy sessions = 219). Specimens were taken for CH, FNAC, and MABS at each biopsy session, but only the result of one technique (previously randomly allocated) was communicated back to the clinical team, using a standardized grading system. Subsequently the presence or absence of rejection was determined by retrospective analysis of the clinical and biochemical course by 4 clinicians, without reference to the biopsy result. Graft survival was not significantly different in the three groups. The sensitivities for CH, FNAC, and MABS were 75%, 59%, and 77%, respectively, while the specificities were 87%, 96%, and 80%, respectively. Inadequate samples for analysis occurred frequently with the MABS technique--and, to a lesser extent, with CH--and both techniques tended to overdiagnose rejection. FNAC most often gave an answer but did miss clinically important rejection episodes. Needle-core biopsy processed for CH remains the most reliable biopsy technique for the diagnosis of rejection of renal allografts. FNAC is a useful technique for monitoring grafts with stable function or nonfunction. MABS does provide information equivalent to CH, but, in this study, had a high incidence of inadequate samples.

Antibodies, Monoclonal↗
PubMed1992

Novel signal transduction pathway mediating endothelium-dependent beta-adrenoceptor vasorelaxation in rat thoracic aorta.

1. Isoprenaline (3 x 10(-8)-10(-5) M), salbutamol (3 x 10(-7)-10(-4) M) and forskolin (3 x 10(-9)-3 x 10(-7) M) relaxed rat isolated thoracic aortic rings contracted with noradrenaline (10(-7) M). Removal of the endothelium from the aortic rings abolished the effect of acetylcholine (10(-6) M) and completely prevented the vascular relaxation induced by isoprenaline, salbutamol or forskolin. 2. The isoprenaline concentration-relaxation curve was shifted in parallel to the right about 10 fold by propranolol (3 x 10(-7) M) with no change in the maximum response, showing that the relaxation was mediated by a beta-adrenoceptor. 3. The inhibitor of nitric oxide synthesis, NG-nitro-L-arginine (L-NOARG; 10(-5) M), shifted the isoprenaline relaxation curve to the right and reduced the maximum response. 4. Isoprenaline (10(-6) M) relaxed noradrenaline-induced tone by approximately 95% and at the same time increased levels of adenosine 3':5'-cyclic monophosphate (cyclic AMP) 4 fold and guanosine 3':5'-cyclic monophosphate (cyclic GMP) 12 fold in the aortic rings. Sodium nitroprusside (3 x 10(-8) M) relaxed noradrenaline-evoked tone by 82% without changing levels of cyclic AMP but raised cyclic GMP 19 fold. 5. Forskolin (10(-7) M) relaxed noradrenaline-induced tone by approximately 41% and, like isoprenaline, increased levels of cyclic AMP (2.5 fold) and cyclic GMP (12 fold) in the aortic rings. 6. Removal of the endothelium abolished the relaxant effects of isoprenaline (10(-6) M) and also the associated accumulation of cyclic AMP and cyclic GMP.7. L-NOARG (10- M) inhibited the relaxant responses and accumulation of cyclic GMP induced by isoprenaline (10-6 M) and forskolin (10- M) without affecting the associated cyclic AMP accumulation.8. It is concluded that, in the rat aorta, isoprenaline acts through a P-adrenoceptor on the endothelium to raise cyclic AMP and that this may, directly or indirectly, release nitric oxide to evoke vascular relaxation via the increase in cyclic GMP. The importance of this novel transduction pathway for cardiovascular regulation remains to be determined.

Albuterol↗
PubMed1992

Human alpha-calcitonin gene-related peptide stimulates adenylate cyclase and guanylate cyclase and relaxes rat thoracic aorta by releasing nitric oxide.

1. The signal transduction pathway for vasorelaxation induced by human alpha-calcitonin gene-related peptide (human alpha-CGRP) was studied in rat thoracic aortic rings preconstricted with noradrenaline (10(-7) M). 2. Vasorelaxation by human alpha-CGRP was inhibited by haemoglobin (10(-6) M) and methylene blue (10(-5) M) but was unaffected by ibuprofen (10(-5) M). 3. Acetylcholine caused a 16 fold increase in levels of guanosine 3':5'-cyclic monophosphate (cyclic GMP) with levels of adenosine 3':5'-cyclic monophosphate (cyclic AMP) being unaltered. Human alpha-CGRP caused a 12 fold increase in levels of cyclic GMP but, in contrast to acetylcholine, evoked a 2.5 fold rise in levels of cyclic AMP. The rises in cyclic nucleotides evoked by human alpha-CGRP and acetylcholine were dependent on the presence of an intact endothelium. 4. NG-nitro-L-arginine (L-NOARG: 10(-5) M), which inhibits nitric oxide synthetase, inhibited the relaxant response to human alpha-CGRP and cyclic GMP accumulation without affecting the cyclic AMP accumulation. 5. The data presented in this paper suggests that human alpha-CGRP relaxes the rat thoracic aorta by releasing nitric oxide and stimulating guanylate cyclase. The stimulation of adenylate cyclase by human alpha-CGRP probably precedes the activation of nitric oxide synthase but could be unrelated to the relaxant response.

Acetylcholine↗
PubMed1992

A new method for quantification of islets by measurement of zinc content.

The ability to quantify the yield of pancreatic islet tissue after isolation is important for interlaboratory comparisons and for the assessment of islet yield prior to clinical transplantation. Because pancreatic islets contain a much higher concentration of zinc than other tissues, we investigated the analysis of zinc as a measure of islet tissue yield. Rat islets of standard diameter 250 microns were handpicked into samples containing 10-80 islets. The zinc content was measured by EAAS and showed a linear correlation with islet number. A zinc binding fluorescent dye, TSQ, was investigated as a way of simplifying the zinc measurement for routine use. Samples of 10-80 islets of 250 microns were sonicated in 3 ml zinc-free water, 0.18 mumol TSQ was added, and the TSQ-zinc fluorescence was measured at 480 nm. A linear correlation was observed. Exocrine contamination up to 50% barely affected the results. Islet zinc content also was shown to be correlated linearly with islet number for freshly isolated human islets. Measurement of zinc by TSQ fluorescence is a rapid, cheap, and objective measure of islet tissue content.

Animals↗
PubMed1991

Pancreatic islet transplantation future prospects.

There is good evidence that the long-term complications of diabetes are caused by poor control of blood sugar, even in patients where intensive glucose monitoring is used to control insulin therapy. Pancreatic transplantation offers the potential for normalisation of glucose metabolism, but at the cost of major surgery and immunosuppression. The possibility of separating the insulin-secreting tissue from the exocrine gland has many attractions. Isolated pancreatic islets are small enough to allow transplantation as free grafts by an injection technique. Furthermore, it may be possible to modify the graft in such a way as to prevent rejection with minimal or no immunosuppression. For pancreatic islet transplantation to become useful in clinical practice it will be necessary to develop efficient techniques for harvesting viable islet tissue in adequate quantities, identify a suitable site for transplantation and prevent rejection. Over the past 20 years experimental models of islet transplantation have proven the potential of this approach, but until recently it has not been possible to translate these experiments into clinical practice. Recently, there have been significant advances in the techniques available for separation of islets from the pancreas of large mammals and man, and recent clinical trials of islet transplantation have shown evidence of short-term function. However, significant problems remain, particularly those of rejection and the maintenance of long-term function, before introduction of clinical islet transplantation as standard therapy for diabetes can be expected.

Animals↗
PubMed1991

In vitro staining of islets of Langerhans for fluorescence-activated cell sorting.

A previously described technique from the author's laboratories for purification of pancreatic islets by fluorescence-activated cell sorting used the dye neutral red (NR) to obtain specific fluorescence of islets sufficient to give a sorting signal. A major drawback with this technique was the need to inject the dye intravascularly before excision of the pancreas. Preliminary investigations showed that NR would produce selective staining of islets by topical application in vitro but only at low concentrations that were insufficient to give fluorescence strong enough for sorting. The chelating agent dithizone (DTZ) produces bright red staining of islets by topical application in vitro. Further studies showed that dithizone-stained islets exhibited moderately strong fluorescence that faded too quickly for reliable sorting. By combining both NR and DTZ staining in vitro, selective fluorescence of islets was obtained that was sufficient to allow efficient sorting. Using the combined DTZ/NR stain the yield of islets obtained by sorting from a single rat pancreas was 569 +/- 72 (n = 16), corresponding to 83% of the islets present in the digest. The mean purity of the preparation, confirmed by histologic examination, was 80%. The viability of the islets was shown to be good both by supravital staining and by the successful correction of streptozotocin diabetes in syngeneic rats following transplantation of sorted islets.

Animals↗
PubMed1990

Mechanism of phenylalanine regulation of phenylalanine hydroxylase.

The mechanism of phenylalanine regulation of rat liver phenylalanine hydroxylase was studied. We show that phenylalanine "activates" phenylalanine hydroxylase, converting it from an inactive to active form, by binding at a true allosteric regulatory site. One phenylalanine molecule binds per enzyme subunit; it remains at this site during catalytic turnover and, while there, cannot be hydroxylated. Loss of phenylalanine from the site causes a loss of enzymatic activity. The rate of loss of activation is dramatically slowed by phenylalanine, which kinetically "traps" activated enzyme during relaxation from the activated to unactivated state. An empirical equation is presented which allows calculation of relaxation rates over a wide range of temperatures and phenylalanine concentrations. Kinetic trapping by phenylalanine is a novel effect. It was analyzed in detail, and its magnitude implied that phenylalanine activation involves cooperativity among all four subunits of the enzyme tetramer. A regulatory model is presented, accounting for the properties of the phenylalanine activation reaction in the forward and reverse directions and at equilibrium. Fluorescence quenching studies confirmed that activation increases the solvent accessibility of the enzyme's tryptophan residues. Physical and kinetic properties of purified phenylalanine hydroxylase from rat, rabbit, baboon, and goose liver were compared. All enzymes were remarkably alike in catalytic and regulatory properties, suggesting that control of this enzyme is similar in mammals and birds.

Animals↗
PubMed1990

Anatomical aspects of the blood supply to the skin of the posterior calf: technique of below-knee amputation.

Whether or not the soleus muscle should be excised during below-knee amputation is a point of contention. The anatomical basis for the blood supply to the skin of the posterior calf has been examined by dissection and selective arterial injection of cadavers. The results showed that the skin used to construct the posterior flap for below-knee amputation derives its blood supply from both axial vessels, running with the peripheral nerves, and perforating vessels arising through the gastrocnemius muscle. There is no contribution by blood vessels that pass through the soleus muscle. When a posterior flap for below-knee amputation is constructed the soleus muscle should be completely excised.

Amputation, Surgical↗
PubMed1990

Islet isolation and transplantation techniques in the primate.

Aspects of islet transplantation in the primate have been investigated using a model of autotransplantation of islets in the monkey after total pancreatectomy. The results showed that the subcapsule of the kidney was not a good site for implantation of relatively impure islet preparations, or of purified islet preparations if the mass of tissue implanted was marginal. The spleen was a much better site for the islet implantation, allowing fasting normoglycemia in many of the animals. The results of histologic examination of the spleen four to six weeks after intrasplenic islet transplantation showed good preservation of islet morphologic features, with numerous islets scattered throughout the spleen. Cells secreting insulin, glucagon and somatostatin were present in approximately normal proportions. Transplantation of islets into the portal vein produced good short and long term function. A number of grafts failed between four and 18 months, this failure being related to the function demonstrated at six weeks by intravenous glucose tolerance tests. Animals with poor initial function tended to fail early. However, those with good initial function continued to gain weight and remain normoglycemic up to 2.5 years, providing encouragement to improve further the yield and purity of the islet preparation as well as seeking more efficient techniques for islet implantation.

Animals↗
PubMed1989

The specificity of rejection and the absence of susceptibility of pancreatic islet beta cells to nonspecific immune destruction in mixed strain islets grafted beneath the renal capsule in the rat.

The specificity of rejection of isolated pancreatic islets was examined in the rat using a quantitative model in which syngeneic (DA) or a mixture of syngeneic and allogeneic (DA and LEW or PVG) islets were implanted beneath the capsule of the kidney of nondiabetic normal rats (DA). 3 wk after transplantation total insulin extraction assays of the kidney with its islet implant together with immunohistological examination of the site of transplantation for evidence of syngeneic or allogeneic tissue demonstrated the total destruction of allogeneic islets without any evidence of damage to syngeneic islets either distant or in immediate proximity to allogeneic islets. Pancreatic islets, and especially beta cells, appear to be particularly vulnerable to the effector arm of both autoimmune and alloimmune responses, a vulnerability that has been attributed to the cytotoxic effects of lymphokines, notably IL-1, released in both autoimmune and alloimmune responses. The experiments reported here demonstrate not only the exquisite specificity of the allograft reaction but are not compatible with a hypothesis that B cells within an intact islet are nonspecifically susceptible to destruction by lymphokines.

Animals↗
PubMed1989

The effect of hyperglycaemia on pancreatic islets transplanted into rats beneath the kidney capsule.

The effect of hyperglycaemia on islet transplantation in the rat has been examined in two ways, using syngeneic transplantation of 400 islets to the kidney capsule and subsequent measurement of kidney insulin content as a measurement of B-cell survival. Firstly, islets were transplanted into either diabetic or normal rats, then 6 months later the composite kidney/islet graft was transplanted into a normal rat. The insulin content was measured after a further 6 months and was found to be significantly reduced in islets exposed to hyperglycaemia in the primary recipient. These findings are interpreted as showing that long-term exposure of islets to hyperglycaemia results in B-cell loss. In the second experiment 400 islets were transplanted into either a long-term (6 months) diabetic or a normal rat. Two weeks later the composite kidney/islet graft was transplanted into a normal rat. The insulin content was measured after a further 6 months and no significant difference was found, whether the primary recipient was diabetic or not. These results are interpreted as showing that islet graft implantation is not impaired in long-term diabetic rats.

Animals↗
PubMed1989

The ATP- and tolbutamide-sensitivity of the ATP-sensitive K-channel from human pancreatic B cells.

The ATP- and sulphonylurea-sensitivity of the ATP-sensitive K-channel was measured in human pancreatic B cells. In inside-out patches, half-maximal inhibition of channel activity was produced by 10 mumol/l ATP (with 2 mM Mg2+) and ATP-inhibition was partially antagonised by ADP. A significantly lower sensitivity to ATP was found in whole-cell recordings. Tolbutamide inhibited whole-cell ATP-sensitive K-currents half-maximally at 18 mumol/l; the sensitivity to tolbutamide was somewhat less in the inside-out patch. Ca-activated K-channels were unaffected by tolbutamide (10 mmol/l). These results resemble those found for rodent B cells and suggest that sulphonylureas exert their therapeutic effects in Type 2 (non-insulin dependent) diabetes by inhibition of the ATP-sensitive K-channel.

Adenosine Triphosphate↗