The Stimulus-Secretion Coupling of Glucose-Induced Insulin Release. VII. A PROPOSED SITE OF ACTION FOR ADENOSINE-3′,5′-CYCLIC MONOPHOSPHATE

Glucose-induced insulin release is thought to result from the following sequence of events in the beta cell: glucose metabolism leading to the production of a metabolic signal, net calcium uptake by the beta cell in response to the signal, and interaction between calcium and a microtubular-microfilamentous system, leading to emiocytosis of the secretory granules. Dibutyryl-cyclic AMP (db-cAMP) and theophylline are known to potentiate glucose-induced insulin release, their insulinotropic action being most marked at high glucose concentrations. Based on the above mentioned concepts, it was considered in the present experiments that the primary site of action of cAMP in the beta cell could correspond to either a facilitation of glucose metabolism, a modification of calcium distribution, or an interaction with the microtubular-microfilamentous system.The first of these hypotheses appeared unlikely because db-cAMP and theophylline, in sharp contrast with other agents known to affect glucose metabolism in the beta cell, did not modify glucose-induced calcium uptake by isolated islets incubated at high glucose concentrations. The last hypothesis also appeared unlikely since theophylline did not interfere with the deleterious effect of colchicine on the microtubular system, and since vincristine or colchicine did not differentially affect the respective insulinotropic action of glucose and theophylline. An effect of cAMP upon calcium distribution in the beta cell was suggested by the following findings. Whereas glucose and leucine were unable to promote insulin release in the absence of extracellular calcium, the addition of db-cAMP or theophylline to the calcium-depleted media partially restored theinsulinotropic action of glucose and leucine. Moreover, theophylline caused a dramatic increase in (45)Ca efflux from perifused islets, even in the absence of glucose. It is concluded that the insulinotropic action of cAMP could be due to a glucose-independent translocation of calcium within the beta cell, from an organelle-bound pool to a cytoplasmic pool of ionized calcium readily available for transport across the cell membrane.     

Can the reading for serologic reactivity following 37 degrees C incubation be omitted?

The need to detect antibodies that agglutinate and/or hemolyze red cells (RBCs) directly at 37 degrees C, but do not react in subsequently performed indirect antiglobulin tests (IATs), is of concern relative to the streamlining and automation of antibody detection methods. To determine incidence and significance of such reactions, data from 87,480 tests, which used low-ionic-strength saline, 10-minute incubation at 37 degrees C, and anti-IgG, were analyzed for unexpected antibodies. There were 3590 positive tests, of which 475 showed reactions at 37 degrees C but not in subsequently performed IATs (37 + IAT-). Of these, 196 reactions were due to autoantibodies or other factors usually considered insignificant with respect to the survival of transfused incompatible RBCs, 176 were due to alloantibodies of questionable clinical significance (M, Lea, P1, etc.), and 103 were associated with alloantibodies of potential clinical significance (63 E, 27 K, 5 Jka, 4 D, 3 cE, and 1 C). This latter reaction was seen in 72 patients, with two 37 + IAT-antibodies occurring in each of 3 patients. Of the 75 potentially significant 37 + IAT-antibodies, 57 were seen in patients recently exposed to homologous RBCs, 13 in patients with a history of transfusion and/or pregnancy, and 5 in patients with no known exposure to homologous RBCs. IAT reactivity was observed in subsequent samples with 27 of these antibodies. The predictive value of a 37 + IAT-test was 21.7 percent for a potentially significant antibody. The incidence was 0.12 percent of all tests for unexpected antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Percutaneous umbilical blood sampling and umbilical vein transfusions. Rapid serologic differentiation of fetal blood from maternal blood

Percutaneous umbilical blood samples (PUBS), obtained under ultrasound guidance, are used for prenatal diagnosis and management of hemolytic disease of the newborn (HDN) and other fetal disorders. Rapid testing at the time of sampling is vital to distinguish fetal from maternal blood. Blood typing was performed by slide technique in the treatment room during 38 procedures on 25 patients. Anti-I was used to test 50 presumed PUBS; venous I-positive maternal blood was tested in parallel. Because anti-I cannot detect fetal blood after umbilical vein transfusion (UVT) of I-positive donor blood, ABO and Rh blood typing reagents were used to test 29 samples when maternal and fetal or donor blood groups differed. Monoclonal reagents were used for optimal detection of weak AB antigens in fetal blood. Avid, chemically modified anti-D was used for Rh typing. Blood typing showed 27 (34%) of 79 samples to be maternal blood. Fetal blood was obtained in 8 of 10 cases investigated for fetal disorder and in 16 cases of potential HDN (anti-D, 5; -CD, 5; -cE, 2; -K, 2; -c; -E). The absence of HDN (antigen-negative fetus) was determined in 4 cases. UVT afforded live birth of 9 of 10 infants with HDN and was not indicated in two cases.     

Stimulation of proinsulin biosynthesis by K+ in rat pancreatic islets

Glucose-stimulated proinsulin biosynthesis is regulated mainly at the translational level. This study aims at investigating the possible role of the B-cell K⁺ content in such a process. In order to increase the islet cells K⁺ content, rat pancreatic islets exposed to a low D-glucose concentration (e.g., 2.5 mM) were incubated in the presence of 30 or 60 mM K⁺, as distinct from a control extracellular K⁺ concentration of 5 mM. Under these conditions, the K⁺ content of the islets, as judged from the net uptake of ⁸⁶Rb⁺ over 60 min incubation, was increased to a level comparable to that otherwise found in the presence of 16.7 mM D-glucose. In the presence of 2.5–4.0 mM D-glucose, the rise in K⁺ concentration from 5 to 30 and 60 mM caused a progressive increase in the incorporation of l-[4-³H]phenylalanine into both all islet peptides and (pro)insulin. A preferential stimulation of proinsulin biosynthesis was only observed in islets incubated at 60 mM K⁺ in the presence of 4.0 mM D-glucose. In relative terms, the K⁺-induced increase in biosynthetic variables was less pronounced, however, than that otherwise evoked by a rise in D-glucose concentration from 2.5 to 4.0 mM to 5.6 or 16.7 mM. These findings may suggest that the effect of D-glucose to increase the K⁺ content of islet cells represents one modality for coupling a rise in D-glucose concentration to stimulation of proinsulin biosynthesis.     

The role of calcium in glucagon release,interactions between glucose and calcium

Summary The interrelationships between glucose and calcium in glucagon release were investigated using the dynamic system of the in vitro perfused rat pancreas. When calcium deprivation was induced in the presence of fixed concentrations of glucose prevailing throughout the experiments (3.3, 5.5, 8.3 and 16.6 mM), an enhancement of glucagon release invariably occurred, the shape and amplitude of such response differing in relation to the environmental glucose concentration. Such enhancement of glucagon release was readily reversible upon restoration of normal calcium levels. By contrast, during the period of calcium deprivation itself, glucagon release was little influenced by either raised (from 3.3 to 16.6 mM) or decreased (from 16.6 to 3.3 mM) glucose concentrations. These results clearly indicate that calcium plays, at least, a dual role — both inhibitory and permissivein glucagon secretion, but the intimate mechanisms by which calcium exerts such a dual action are at present unknown.     

Internalization of glimepiride and glibenclamide in the pancreatic B-cell

When rat pancreatic islets were exposed for 30 min at 37°C to tritiated glibenclamide (2.5 M) or glimepiride (1.5 M), internalization of these hypoglycaemic sulphonylureas in B and non-B cells was observed by autoradiography. In the B cell, the autoradiographic grains were often associated with secretory granules.     

Is the glucose-induced phosphate flush in pancreatic islets attributable to gating of volume-sensitive anion channels?

d-Glucose and other nutrient insulin secretagogues have long been known to induce a transient increase in inorganic phosphate release from pancreatic islets, a phenomenon currently referred to as a “phosphate flush”. The objective of this study was to explore the possible participation of volume-sensitive anion channels in such a process. Rat pancreatic islets were preincubated for 60 min in the presence of [³²P]orthophosphate and then perifused for 90 min to measure ³²P fractional outflow rate and insulin secretion. From minutes 46 to 70 inclusive either the concentration of d-glucose was increased from 1.1 to 8.3 mmol L⁻¹ or the extracellular osmolarity was decreased by reducing the NaCl concentration by 50 mmol L⁻¹. The increase in d-glucose concentration induced a typical phosphate flush and biphasic stimulation of insulin release. Extracellular hypoosmolarity caused a monophasic increase in both effluent radioactivity and␣insulin output. The inhibitor of volume-sensitive anion␣channels 5-nitro-2-(3-phenylpropylamino)benzoate (0.1 mmol L⁻¹) inhibited both stimulation of insulin release and phosphate flush induced by either the increase in d-glucose concentration or extracellular hypoosmolarity. It is proposed that gating of volume-sensitive anion channels accounts for the occurrence of the phosphate flush and subsequent stimulation of insulin secretion in response to either an increase in d-glucose concentration or a decrease in extracellular osmolarity.     

Reducing Diagnostic Errors in Musculoskeletal Trauma by Reviewing Non-Admission Orthopaedic Referrals in the Next-Day Trauma Meeting

INTRODUCTION

Diagnostic errors in orthopaedics are usually caused by missing a fracture or misreading radiographs. The aim of this study was to document the pick-up rate of the wrong diagnoses by reviewing X-rays and casualty notes in the next-day trauma meeting.

PATIENTS AND METHODS

The casualty notes and radiographs of 503 patients were prospectively reviewed in the daily trauma meeting between August 2002 and December 2002 in a district general hospital. The relevant data were collected and analysed by a single assessor.

RESULTS

The false positive rate for making an orthopaedic diagnosis was 12.6% (i.e.) diagnosing a fracture, when none existed). The false negative (missing) rate was 4%, while 2.4% incidental findings were missed, or at least not documented, after reading the X-rays. There were 7.8% wrong diagnoses made. The majority of the patients were seen by the senior house officers.

CONCLUSIONS

The medicolegal significance of false negative diagnosis is obviously greater. In a busy emergency department, where a large number of patients are seen, there is a greater risk. This study shows the importance in a small-to-medium sized accident and emergency unit as well, where there is no senior cover available out-of-hours for final radiological interpretation. A morning trauma meeting which covers reviewing admitted patients as well as non-admission orthopaedic referrals has an effective risk management solution to early detection of missed and wrong diagnoses.