A conducting salt-based amperometric biosensor for measurement of extracellular lactate accumulation in ischemic myocardium

In this paper, fabrication, characterization, and physiological application of a miniaturized amperometric lactate biosensor are described. The sensor is based on cross-linked lactate oxidase and tetrathiafulvalene-tetracyano-quinodimethane (TTF-TCNQ) charge transfer complex. The sensor was developed for continuous quantitative measurement of the lactate accumulation in ischemic myocardium under severe depletion of oxygen. The sensor was evaluated in vitro at an applied potential of 0.15 V vs Ag/AgCl; it proved to combine all the performance characteristics desired for the present application, such as proper response in absence of oxygen, good operational stability, good accuracy and precision (103.5 +/- 1.2%), adequate response time (t95% = 80 s), and wide linear dynamic range up to 27 mM (r = 0.9998) in N2-saturated solutions and at 37 degrees C. The prepared sensors (n = 12) showed sensitivity of 380 +/- 90 nA/mM, and a background current of 240 +/- 50 nA. The lower limit of detection is 0.4 +/- 0.15 mM with a S/N ratio equal to 3. Results obtained for direct lactate monitoring in ischemic rabbit papillary muscle under no-flow conditions and PO2 < 6 mm Hg are presented.     

Accuracy of the one-point in vivo calibration of "wired" glucose oxidase electrodes implanted in jugular veins of rats in periods of rapid rise and decline of the glucose concentration

The hypothesis of the feasibility of one-point in vivo calibration of intravenously implanted glucose sensors during periods of rapid rise and decline of venous blood glucose concentration was tested. Miniature (5 x 10(-4) cm2 mass transporting area) glucose electrodes with 10-90% response times < 2 min, that did not consume oxygen, were implanted in jugular veins of systemically heparinized rats and used in 4-h experiments, during which the blood glucose concentration was amperometrically monitored. The glucose electrodes were made by electrically connecting ("wiring") reaction centers of glucose oxidase through an electron-conducting redox hydrogel to gold electrode surfaces. The redox polymer and enzyme constituting the electrode sensing layer were immobilized by cross-linking, and thus the electrodes had no diffusional and readily leached redox mediator. One hour after their implantation, the electrodes accurately tracked the blood glucose concentration when calibrated in vivo by a one-point calibration, when the glucose concentration was steady, when rising rapidly, and when declining steeply. For an assumed 2-min lag time, the sensor readings were well correlated with the true blood glucose concentrations, with linear regression analysis yielding a slope of 0.97 +/- 0.07 and an intercept (bias) of 0.3 +/- 0.3 mM. The correlation coefficient, r2, was 0.949 +/- 0.020, and the percent difference through the 2-22 mM range was 1.9 +/- 1.0%. The results suggest that, in combination with understanding and modeling of transient physiological differences between the subcutaneous and the blood glucose concentrations, it will be possible to calibrate by one-point in vivo calibration subcutaneously implanted sensors, even while the glucose concentration changes rapidly.     

High-throughput flow-injection analysis of glucose and glutamate in food and biological samples by using enzyme/polyion complex-bilayer membrane-based electrodes as the detectors

The concentration of glucose was determined by a combination of flow injection analysis (FIA) with amperometric enzyme sensor detection. The enzyme sensor was prepared by immobilizing glucose oxidase on an electrode coated with a polyion complex layer consisting of poly-L-lysine and poly(4-styrenesulfonate). The inner, polyion complex layer was useful for preventing electrochemical interferents (e.g., L-ascorbic acid, uric acid and acetaminophen) from reaching the electrode surface, which was effective for reducing the interferential responses upon the injections of biological and food samples. The sensor-based system could be used for the determination of glucose from 10 microM to 3 mM with the sampling rate of 180 h-1, and was stable for more than 2 months. An FIA system for determining L-glutamic acid (3 microM-0.5 mM) was also prepared by using an enzyme electrode based on a glutamate oxidase/polyion complex-bilayer as the detector.     

Simultaneous determination of maltose and glucose using a screen-printed electrode system

A screen-printed sensor system consisting of a glucose oxidase (GOD) electrode and an amyloglucosidase/glucose oxidase (A/G) electrode was constructed to determine maltose and glucose simultaneously in a mixture. Sensor construction was optimised so that it contained 20 units of GOD/40 units of amyloglucosidase and 0.2 mM 1,1'-ferrocenedimethanol. These components were deposited onto a screen-printed carbon electrode and an outer membrane was printed from 3.5% hydroxyethyl cellulose (HEC) solution. The optimum pH was 4.8. The linear range of the system was up to 40 mM glucose or 20 mmol/L maltose with coefficients of variation (CVs) ranging from 3.5% to 5.29%. The results obtained by using the enzyme electrode system agreed well with those obtained by the Fehling titration method. When stored dry, especially at 4 degrees C, the enzyme electrodes showed good stability over four months.     

Alterations in glucose metabolism in the elderly patient with diabetes

OBJECTIVE: To determine the alterations in glucose metabolism in elderly patients with NIDDM. RESEARCH DESIGN AND METHODS: We studied 9 healthy elderly control subjects (73 +/- 1 yr of age; body mass index 25.7 +/- 0.4 kg/m2) and 9 untreated elderly NIDDM patients (72 +/- 2 yr of age; BMI 25.9 +/- 0.5 kg/m2). Each subject underwent a 3-h oral glucose tolerance test (40 g/m2); a 2-h hyperglycemic glucose clamp study (glucose 5.4 mM above basal); and a 4-h euglycemic insulin clamp (40 mM.m2.min-1). Tritiated glucose methodology was used to measure glucose production and disposal rates during the euglycemic clamp. RESULTS: Patients with NIDDM had a higher fasting glucose (9.3 +/- 0.3 vs. 5.1 +/- 0.1 mM in control subjects vs. NIDDM patients, respectively, P < 0.001) and a greater area under the curve for glucose during the OGTT (16.0 +/- 0.6 vs. 6.7 +/- 0.3 mM in control subjects vs. NIDDM patients, respectively, P < 0.01) than the healthy control subjects. During the hyperglycemic clamp, patients with NIDDM had an absent first-phase insulin response (112 +/- 6 vs. 250 +/- 31 pM in control subjects vs. NIDDM patients, respectively, P < 0.01), and a blunted second-phase insulin response (159 +/- 11 vs. 337 +/- 46 pM in control subjects vs. NIDDM patients, respectively, P < 0.01). Before the euglycemic clamp, fasting insulin (99 +/- 5 vs. 111 +/- 10 pM in control subjects vs. NIDDM patients, respectively) and hepatic glucose production (11.8 +/- 0.7 vs. 11.5 +/- 0.5 mumol.kg-1-min-1 in control subjects vs. NIDDM patients, respectively) were similar. Steady-state (180-240 min) glucose disposal rates during the euglycemic clamp were slightly, but not significantly, higher in the normal control subjects (36.5 +/- 1.1 vs. 33.1 +/- 1.9 mumol.kg-1-min-1 in control subjects vs. NIDDM patients, respectively, NS). CONCLUSIONS: We conclude that NIDDM in nonobese elderly subjects is characterized by a marked impairment in insulin release. This may be attributable to the toxic effects of chronic hyperglycemia on the beta-cell. When compared with age-matched control subjects, the NIDDM patients showed no increase in fasting insulin or hepatic glucose production, and insulin resistance was mild.     

Intraneuronal [Ca2+] changes induced by 2-deoxy-D-glucose in rat hippocampal slices

Temporary replacement of glucose by 2-deoxyglucose (2-DG; but not sucrose) is followed by long-term potentiation of CA1 synaptic transmission (2-DG LTP), which is Ca2+-dependent and is prevented by dantrolene or N-methyl--aspartate (NMDA) antagonists. To clarify the mechanism of action of 2-DG, we monitored [Ca2+]i while replacing glucose with 2-DG or sucrose. In slices (from Wistar rats) kept submerged at 30 degreesC, pyramidal neurons were loaded with [Ca2+]-sensitive fluo-3 or Fura Red. The fluorescence was measured with a confocal microscope. Bath applications of 10 mM 2-DG (replacing glucose for 15 +/- 0.38 min, means +/- SE) led to a rapid but reversible rise in fluo-3 fluorescence (or drop of Fura Red fluorescence); the peak increase of fluo-3 fluorescence (DeltaF/F0), measured near the end of 2-DG applications, was by 245 +/- 50% (n = 32). Isosmolar sucrose (for 15-40 min) had a smaller but significant effect (DeltaF/F0 = 94 +/- 14%, n = 10). The 2-DG-induced DeltaF/F0 was greatly reduced (to 35 +/- 15%, n = 16) by,-aminophosphono-valerate (50-100 microM) and abolished by 10 microM dantrolene (-4.0 +/- 2.9%, n = 11). A substantial, although smaller effect, of 2-DG persisted in Ca2+-free 1 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N', N'-tetraacetic acid (EGTA) medium. Two adenosine antagonists, which do not prevent 2-DG LTP, were also tested; 2-DG-induced DeltaF/F0 (fluo-3) was not affected by the A1 antagonist 8-cyclopentyl-3, 7-dihydro-1,3-dipropyl-1H-purine-2,6-dione (DPCPX 50 nM; 287 +/- 38%; n = 20), but it was abolished by the A1/A2 antagonist 8-SPT; 25 +/- 29%, n = 19). These observations suggest that 2-DG releases glutamate and adenosine and that the rise in [Ca2+] may be triggered by a synergistic action of glutamate (acting via NMDA receptors) and adenosine (acting via A2b receptors) resulting in Ca2+ release from a dantrolene-sensitive store. The discrepant effects of sucrose and 8-SPT on DeltaF/F0, on the one hand, and 2-DG LTP, on the other, support other evidence that increases in postsynaptic [Ca2+]i are not essential for 2-DG LTP.     

Lack of nephrotoxicity of oral ammine/amine platinum (IV) dicarboxylate complexes in rodents

The comparative nephrotoxicity of i.v. cisplatin, i.v. carboplatin and six p.o. ammine/amine Pt(IV) dicarboxylates was studied in rodents following single MTD treatments. In mice, i.v. cisplatin caused proteinuria (1 g l-1), glycosuria (16.7 mM) and decreased GFR at 4 days, and histological kidney damage with onset at 6 days. In contrast, mice treated with i.v. carboplatin or p.o. ammine/amine Pt(IV) dicarboxylates had urinary glucose, urinary protein, GFR and kidney histology within the control range. In rats, i.v. cisplatin caused 5-fold elevations in plasma creatinine (188 +/- 33 microM) and urea (30.4 +/- 8.9 mM), a 10-fold fall in creatinine clearance (0.54 +/- 0.31 ml min-1 kg-1), a 25-fold elevation in urine/plasma glucose concentration ratio (3.28 +/- 0.17), a 20% increase in kidney weight (7.9 +/- 0.56 mg gm-1 body weight) and extensive histological damage 4 days after treatment. In contrast, i.v. carboplatin and p.o. JM216 (the lead compound of this series) caused neither abnormalities in renal function nor histological damage in rats. The nephrotoxicity of single MTD treatments of p.o. ammine/amine Pt(IV) dicarboxylate complexes appears less than i.v. cisplatin and comparable to i.v. carboplatin.     

Vasopressin response to 2-deoxy-D-glucose in the rat

The effect of 2-deoxyglucose (2DG) upon plasma arginine vasopressin (AVP), measured by a specific and sensitive RIA, has been studied in unanesthetized male Sprague-Dawley rats. Injection of the drug ip at a dose of 100 mg/kg BW caused a 10-fold rise in plasma AVP to 23.4 +/- 4.9 pg/ml (mean +/- SD). This effect was associated with a rise in plasmas osmolality to 301.8 +/- 3.9 mosmol/kg, but this change was accounted for totally by a rise in plasma glucose to 17.3 +/- 3.0 mM and in plasma urea to 3.4 +/- 0.4 mM. The only effective solute, plasma sodium, fell to 135.0 +/- 0.8 mM. Idiogenic osmoles did not change. Blood volume was reduced by about 5%, while PRA and mean arterial pressure did not change. Thus, the rise in plasma AVP caused by 2DG was not mediated by osmotic or hemodynamic stimuli. Treatment with propranolol (2.5 mg/kg BW) 15 min before 2DG completely abolished the AVP response. These results indicate that 2DG stimulates AVP release by a primary mechanism different from that which mediates the effect of insulin-induced hypoglycemia on AVP.     

Kinetic uniformity of the ATP hydrolysis reaction catalyzed by Mg2+-ATPase in smooth muscle cell membrane

The kinetic properties of Mg(2+)-ATPase (EC 3.6.1.3) from myometrium cell plasma membranes have been studied. Under conditions of enzyme saturation with ATP (0.5-1.0 mM) or Mg2+ (1.0-5.0 mM) the initial maximal rates of the Mg(2+)-dependent enzymatic ATP hydrolysis, V0 ATP and V0 Mg, are 27.4 +/- 3.3 and 25.2 +/- 4.1 mumol Pi/hour/mg of protein, respectively. The apparent Michaelis constant, Km, for ATP and of the apparent activation constant, K alpha, for Mg2+ are equal to 28.1 +/- 2.6 and 107.0 +/- 26.0 microM, respectively. The bivalent metal ions used at 1.0 mM suppress the Mg(2+)-dependent hydrolysis of ATP whose efficiency decreases in the following order: Cu2+ > Zn2+ = Ni2+ > Mn2+ > Ca2+ > Co2+. Alkalinization of the incubation medium from pH 6.0 to pH 8.0 stimulates the Mg(2+)-dependent hydrolysis of ATP. It has been found that Mg(2+)-ATPase has the properties of an H(+)-sensitive enzymatic sensor which is characterized by a linear dependence between the initial maximal rate of the reaction, V0, and the pH value. The feasible role of plasma membrane Mg(2+)-ATPase in some reactions responsible for the control of proton and Ca2+ homeostasis in myometrium cells has been investigated.     

Granulosa cells have calcium-dependent action potentials and a calcium-dependent chloride conductance

We have found chicken granulosa cells to be excitable. Experiments using the whole-cell patch-clamp technique showed that they had membrane resting potentials of -62 +/- 3 mV (n = 8) and generated action potentials, either in response to 10-ms depolarizing current pulses or, on occasion, spontaneously. The action potentials persisted in a Na(+)-free bath and were reversibly blocked by 4 mM Co2+. They lasted 0.9-3.0s with 64 mM Cl- in the pipette, were shortened 67 +/- 8% by the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 20 microM), and lengthened to 8.7 +/- 2.2 when the Cl- equilibrium potential (Vcl) was changed from -20 mV to -2 mV by using 134 mM Cl- in the pipette. With conventional whole-cell voltage-clamp, slowly activating and inactivating currents, which reached maximum amplitude after 0.35-1.40 s, were evoked by depolarizing voltage steps. These slow currents activated between voltage steps of -60 mV and -50 mV and reached a maximum inward amplitude at about -40 mV. Changing the Cl- concentration in the pipette (VCl of -2MV or -20 mV) or bath (VCl of -2 mV or + 18 mV) shifted their reversal potential in a direction consistent with a Cl- electrode. They were inhibited by the Cl- channel antagonists 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS; 0.5 mM), NPPB (20 microM), and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS; 0.5 mM). The slow currents were blocked by Ca2+ deprivation, or by CO2+ (4 mM), or by replacing external Ca2+ with Ba2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

A fractally coated, 1.3 mm2 high impedance pacing electrode

Minimizing the geometric surface area of pacing electrodes increases impedance and reduces the current drain during stimulation, provided that voltage (pulse-width) thresholds remain unchanged. This may be feasible by coating the electrode surface to increase the capacity of the electrode tissue interface and to diminish polarization. Ten unipolar, tined leads with a surface area of 1.3 mm2 and a "fractal" coating of iridium (Biotronik SD-V137) were implanted in the ventricle, and electrogram amplitude (unfiltered), slew-rate, pacing threshold (0.5 ms), and impedance (2.5 V; 0.5 ms) were measured by the 5311 PSA (Medtronic). On days 0. 2. 5. 10, 28, 90, 180, 360 postimplant, sensing threshold (up to 7.0 mV, measuring range 1-14 mV on day 360 only) and the strength duration curve (0.5-4.0 V; 0.03-1.5 ms; steps: 0.5 V; 0.01 ms, respectively) were determined, the minimum charge delivered per pulse (charge threshold), and the impedance were taken from pacemaker telemetry (Intermedics 294-03). Data were compared with those of an earlier series of 20 unipolar, tined TIR-leads (Biotronik) with a surface area of 10 mm2 and a @actal" coating of titanium nitride. With the model SD-V137 versus TIR, intraoperative electrogram amplitudes were 15.1 +/- 6.1 versus 14.4 +/- 3.9 mV (NS), slew rates 3.45 +/- 1.57 versus 1.94 +/- 1.06 V/s (P < 0.05), pacing thresholds 0.16 +/- 0.05 versus 0.52 +/- 0.15 V (P < 0.01) and impedance measurements 1,136 +/- 175 versus 441 +/- 73 omega (P < 0.0001), respectively. During follow-up, sensing thresholds were the same with both leads. Differences in pulse width thresholds lost its significance on day 28 but resumed on day 360 (SD-V137; 0.08 +/- 0.04 ms; TIR: 0.16 +/- 0.06 ms at 2.5 V; P < 0.01). With an electrode surface of 1.3 mm2, charge per pulse and impedance consistently differed from control, being 0.15 +/- versus 0.66 +/- 0.20 microC (P < 0.001) and 1,344 +/- 376 versus 538 +/- 79 omega respectively, one year after implantation (P < 0.0001). In summary, "fractally" coated small surface electrodes do not compromise sensing; by more than doubling impedance against controls they offer pacing thresholds (mainly in terms of charge) that are significantly lower than with the reference electrode.     

Tissue specific expression of FMR-1 provides evidence for a functional role in fragile X syndrome

Prostaglandin E2 levels in isolated rat islets were increased from 64 +/- 11 pg/30 islets when incubated in medium containing 2 mM glucose to 115 +/- 9 pg/30 islets in medium containing 20 mM glucose. In contrast, glyceraldehyde (10 mM) reduced prostaglandin E2 levels to 29 +/- 6 pg/30 islets. Inhibition of glucose metabolism by mannoheptulose (10 mM) abolished the stimulatory effect of glucose on prostaglandin E2 levels and inhibited glucose-induced insulin release. The cyclooxygenase inhibitor, flurbiprofen (20 microM), did not affect insulin release caused by glucose or glyceraldehyde. In the presence of 1 mg/ml bovine serum albumin, insulin secretion induced by 20 mM glucose (6.9 +/- 1.1% of islet insulin content) was reduced by the lipoxygenase inhibitor BW755 C (20 microM) to 3.1 +/- 0.6%, and by the phospholipase A2 inhibitor, p-bromophenacyl bromide (10 microM), to 2.1 +/- 0.8%. In the absence of bovine serum albumin the inhibitory action of BW755 C and p-bromophenacyl bromide on glucose-induced insulin release was significantly more pronounced. These drugs whether in the presence or absence of bovine serum albumin, did not affect glyceraldehyde-stimulated insulin secretion. Glyceraldehyde (10 mM), potentiated glucose-induced insulin release in the presence of 2-8 mM glucose, but not for 10-20 mM glucose. Although the phospholipase A2 activator, melittin, initiated insulin release in the presence of 2 mM glucose and enhanced 10 mM glyceraldehyde-stimulated insulin secretion it had no effect on 20 mM glucose-induced insulin release. These two stimulatory effects of melittin on insulin release were totally abolished by p-bromophenacyl bromide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Initial evaluation of a 290-microm diameter subcutaneous glucose sensor: glucose monitoring with a biocompatible, flexible-wire, enzyme-based amperometric microsensor in diabetic and nondiabetic humans

Results of the initial clinical evaluation in 20 human subjects of a subcutaneously implanted microsensor-based amperometrically glycemia-monitoring system, carried out between April 1994 and June 1995, are reported. The system was based on the electrical connection ("wiring") of the reaction centers of glucose oxidase to a gold electrode and on elimination of the chemicals that interfere with glucose monitoring through their horseradish peroxidase-catalyzed oxidation by internally generated hydrogen peroxide. The sensor was finer than a 29-gauge needle and had no leachable components. Because of its high selectivity for glucose, the sensor output was virtually nil at zero glucose level. This enables prompt "one-point" in vivo calibration of the sensor with a single blood glucose sample. Microsensors were subcutaneously implanted in ten nondiabetic and ten insulin-dependent diabetes mellitus (IDDM) volunteers. All subjects underwent standard meal tests and intravenous glucose-tolerance tests (IVGTT) in addition to hourly plasma glucose measurements. The sensor signals were continuously recorded, and the glucose concentration estimates were derived by calibrating the sensor using a single blood sample (one-point calibration). Regression analysis revealed that the sensor-estimated glucose concentrations were linearly related to the plasma glucose concentrations (r2 = 0.75) over a wide glucose concentration range (2-28 mmol/L) (sensor estimate = plasma 0.96 + 0.26 mmol/L). The difference between the estimated and actual glucose concentration was -0.13+/-0.23 mmol/L [mean +/-95% confidence interval (CI), n = 546], and 95% of the estimates fell in clinically acceptable zones of the Clarke error grid. The sensing delay time was 10.4+/-2.3 min as measured by the IVGTT. The subjects reported no discomfort associated with wearing the sensors.     

Glucose-induced intracellular ion changes in sugar-sensitive hypothalamic neurons

In the lateral hypothalamic area (LHA) of rat brain, approximately 30% of cells showed sensitivity to small changes in local concentrations of glucose. These "glucose-sensitive" neurons demonstrated four types of behavior, three of which probably represent segments of a continuous spectrum of recruitment in response to ever more severe changes in blood sugar. Type I cells showed maximum activity 相似文献   

Drug metabolism biosensors: electrochemical reactivities of cytochrome P450cam immobilised in synthetic vesicular systems

Biosensors containing cytochrome P450cam in a didodecyldimethylammonium bromide vesicular system were prepared by cross-linking onto a glassy carbon electrode (GCE) with glutaraldehyde in the presence of bovine serum albumin. Cyclic voltammetric responses of the sensor in air-free buffer solution showed that the sensor exhibited reversible electrochemistry due to direct electron exchange between the haem Fe(3+/2+) redox system and the GCE surface. In air-saturated solution containing camphor, the biosensor gave an irreversible electrocatalytic current which is compatible with the monooxygenation of the substrate. Steady state amperometric experiments with camphor, adamantanone and fenchone were performed with a biosensor prepared by cross-linking P450cam with glutaraldehyde onto a Pt disc electrode. The sensor was characterised by fast amperometric responses, attaining steady-state in about 20 s in a cobalt sepulchrate mediated electrochemical system. The kinetic parameters of the biosensor were analysed using the electrochemical Michaelis Menten equation. The estimated apparent Michaelis-Menten constant, Km, values for the biosensors were in the range of 1.41-3.9 mM.     

Calcium induced contracture stimulates Na,K-pump rate in isolated sheep cardiac Purkinje fibers

By keeping intracellular Na+ (aiNa) low, the Na,K-pump can prevent Ca2+ overload of cardiomyocytes. We therefore examined whether Ca2+ stimulates Na,K-pump activity in sheep cardiac Purkinje fibers. By removing Ca2+, Mg2+ and K+, the fibers depolarized and aiNa rose to 70 mM. After addition of 6 mM Mg2+ and lowering extracellular Na2+ to 29 mM, 30mM Rb+ was added, and over 10-15 min aiNa recovered to 3-7 mM. Two load-recovery cycles were conducted in 10 fibers. During one of the cycles Ca2+ (0.1-1.0 mM) was added before Rb+, causing a contracture. During recovery aiNa fell faster during Ca2+ contracture than in control cycles. Between 30 and 20 mM the rates were -10.0+/-1.6 and -5.4+/-0.6 mM/min, respectively (P<0.05). In Ca2+-exposed fibers tension fell almost parallel with aiNa. Na, K-pump reactivation caused membrane potential (Vm) to hyperpolarize transiently to -70 mV. Ca2+ did not affect membrane conductance. For a given aiNa during reactivation, Vm was more negative during Ca2+ contracture and depolarized faster (P<0.05). Intracellular pH (pHi) fell from 7.11+/-0.05 to 6.92+/-0.08 (n.s.) during control load-recovery cycles and was 6.83+/-0.14 at the end of the Ca2+ cycles. ATP content of the fibers did not change significantly through two complete load-recovery cycles, but creatine phosphate (CrP) fell by about 40%. By fitting the data to a model incorporating the Hill equation we show that during Ca2+-induced contracture maximum Na,K-pump rate (Vmax) was increased by about 40% and aiNa that causes 50% pump activation (k0.5) was lowered from 21. 2+/-1.6 to 15.5+/-1.4 mM.     

Characterization of cellular defects of insulin action in type 2 (non-insulin-dependent) diabetes mellitus

Seven non-insulin-dependent diabetes mellitus (NIDDM) patients participated in three clamp studies performed with [3-3H]- and [U-14C]glucose and indirect calorimetry: study I, euglycemic (5.2 +/- 0.1 mM) insulin (269 +/- 39 pM) clamp; study II, hyperglycemic (14.9 +/- 1.2 mM) insulin (259 +/- 19 pM) clamp; study III, euglycemic (5.5 +/- 0.3 mM) hyperinsulinemic (1650 +/- 529 pM) clamp. Seven control subjects received a euglycemic (5.1 +/- 0.2 mM) insulin (258 +/- 24 pM) clamp. Glycolysis and glucose oxidation were quantitated from the rate of appearance of 3H2O and 14CO2; glycogen synthesis was calculated as the difference between body glucose disposal and glycolysis. In study I, glucose uptake was decreased by 54% in NIDDM vs. controls. Glycolysis, glycogen synthesis, and glucose oxidation were reduced in NIDDM patients (P < 0.05-0.001). Nonoxidative glycolysis and lipid oxidation were higher. In studies II and III, glucose uptake in NIDDM was equal to controls (40.7 +/- 2.1 and 40.7 +/- 1.7 mumol/min.kg fat-free mass, respectively). In study II, glycolysis, but not glucose oxidation, was normal (P < 0.01 vs. controls). Nonoxidative glycolysis remained higher (P < 0.05). Glycogen deposition increased (P < 0.05 vs. study I), and lipid oxidation remained higher (P < 0.01). In study III, hyperinsulinemia normalized glycogen formation, glycolysis, and lipid oxidation but did not normalize the elevated nonoxidative glycolysis or the decreased glucose oxidation. Lipid oxidation and glycolysis (r = -0.65; P < 0.01), and glucose oxidation (r = -0.75; P < 0.01) were inversely correlated. In conclusion, in NIDDM: (a) insulin resistance involves glycolysis, glycogen synthesis, and glucose oxidation; (b) hyperglycemia and hyperinsulinemia can normalize total body glucose uptake; (c) marked hyperinsulinemia normalizes glycogen synthesis and total flux through glycolysis, but does not restore a normal distribution between oxidation and nonoxidative glycolysis; (d) hyperglycemia cannot overcome the defects in glucose oxidation and nonoxidative glycolysis; (e) lipid oxidation is elevated and is suppressed only with hyperinsulinemia.     

Guanidinium-based potentiometric SO2 gas sensor

An SO2 gas sensor was developed by using a hydrogen sulfite-selective electrode positioned behind a gas-permeable membrane (GPM). The hydrogen sulfite-selective electrode was prepared by incorporating a multicyclic guanidinium ionophore in a plasticized poly(vinyl chloride) membrane. This gas sensor presents important advantages over the conventional Severinghaus-type SO2 gas sensor that contains a pH electrode immersed in an internal solution behind the GPM. The Severinghaus gas sensor suffers interferences from weak acids that can cross the GPM as gases and change the pH of the internal solution. In contrast, in the proposed sensor, the excellent selectivity of the HSO3- electrode and the ability of the GPM to discriminate gaseous from nongaseous species combine to generate the most selective potentiometric SO2 gas sensor reported to date.     

Glucose stimulates renin secretion via adrenergic mechanisms in the rat

To elucidate whether and why glucose directly influences renin secretion, the effect of glucose on renin secretion was investigated in the rat. In an in vivo study, renin activity significantly (p<0.01) increased from the basal value of 7.6 +/- 1.4 to 14.2 +/- 3.2 ng Ang I/ml/hr (mean +/- SD) after intravenous glucose (1.0 g/kg, in 50% glucose solution ) injection. Propranolol (10.5 mg/kg) pretreatment partly abolished the increase in renin activity induced by glucose injection. In an in vitro study, the isolated kidneys of male Wistar rats (200-250 g) were perfused with a basal perfusing medium containing 5.5 mM glucose for 20 min, and then perfused with the medium containing 16.5 mM glucose, 27.5 mM glucose, 5.5 mM glucose + 22 mM mannitol, 27.5 mM glucose + 1 microM phentolamine, or 27.5 mM glucose + 1 microM propranolol for 10 min, respectively. Renin activity was significantly increased from a basal value of 8.1 +/- 4.5 to peak value of 17.9 +/- 3.0 ng Ang I/ml/hr (p<0.01) by 16.5 mM glucose, to 59.0 +/- 10.5 ng Ang I/ml/hr (p<0.005) by 27.5 mM glucose, and to 24.7 +/- 5.8 ng Ang I/ml/hr (p<0.01) by 5.5 mM glucose + 22 mM mannitol. The increase in renin activity in the kidney perfused with 27.5 mM glucose was significantly (p<0.005) higher than that with 16.5 mM glucose or that with 5.5 mM glucose + 22 mM mannitol. The 27.5 mM glucose-stimulated increase in renin activity was not changed by the addition of 1 microM phentolamine, while it was completely abolished by the addition of 1 microM propranolol. These results suggest that glucose has a direct stimulating effect on renin secretion probably through beta-adrenergic mechanisms in the rat.     

Synergistic effect of glucose and prolactin on GLUT2 expression in cultured neonatal rat islets

We studied the synergistic effect of glucose and prolactin (PRL) on insulin secretion and GLUT2 expression in cultured neonatal rat islets. After 7 days in culture, basal insulin secretion (2.8 mM glucose) was similar in control and PRL-treated islets (1.84 +/- 0.06% and 2.08 +/- 0.07% of the islet insulin content, respectively). At 5.6 and 22 mM glucose, insulin secretion was significantly higher in PRL-treated than in control islets, achieving 1.38 +/- 0.15% and 3.09 +/- 0.21% of the islet insulin content in control and 2.43 +/- 0.16% and 4.31 +/- 0.24% of the islet insulin content in PRL-treated islets, respectively. The expression of the glucose transporter GLUT2 in B-cell membranes was dose-dependently increased by exposure of the islet to increasing glucose concentrations. This effect was potentiated in islets cultured for 7 days in the presence of 2 micrograms/ml PRL. At 5.6 and 10 mM glucose, the increase in GLUT2 expression in PRL-treated islets was 75% and 150% higher than that registered in the respective control. The data presented here indicate that insulin secretion, induced by different concentrations of glucose, correlates well with the expression of the B-cell-specific glucose transporter GLUT2 in pancreatic islets.