Effects of muscarinic receptor stimulation on Ca2+ transient, cAMP production and pacemaker frequency of rabbit sinoatrial node cells

We investigated the contribution of the intracellular calcium (Ca _i ²⁺ ) transient to acetylcholine (ACh)-mediated reduction of pacemaker frequency and cAMP content in rabbit sinoatrial nodal (SAN) cells. Action potentials (whole cell perforated patch clamp) and Ca _i ²⁺ transients (Indo-1 fluorescence) were recorded from single isolated rabbit SAN cells, whereas intracellular cAMP content was measured in SAN cell suspensions using a cAMP assay (LANCE^®). Our data show that the Ca _i ²⁺ transient, like the hyperpolarization-activated “funny current” (I _f) and the ACh-sensitive potassium current (I _K,ACh), is an important determinant of ACh-mediated pacemaker slowing. When I _f and I _K,ACh were both inhibited, by cesium (2 mM) and tertiapin (100 nM), respectively, 1 μM ACh was still able to reduce pacemaker frequency by 72%. In these I _f and I _K,ACh-inhibited SAN cells, good correlations were found between the ACh-mediated change in interbeat interval and the ACh-mediated change in Ca _i ²⁺ transient decay (r ² = 0.98) and slow diastolic Ca _i ²⁺ rise (r ² = 0.73). Inhibition of the Ca _i ²⁺ transient by ryanodine (3 μM) or BAPTA-AM (5 μM) facilitated ACh-mediated pacemaker slowing. Furthermore, ACh depressed the Ca _i ²⁺ transient and reduced the sarcoplasmic reticulum (SR) Ca²⁺ content, all in a concentration-dependent fashion. At 1 μM ACh, the spontaneous activity and Ca _i ²⁺ transient were abolished, but completely recovered when cAMP production was stimulated by forskolin (10 μM) and I _K,ACh was inhibited by tertiapin (100 nM). Also, inhibition of the Ca _i ²⁺ transient by ryanodine (3 μM) or BAPTA-AM (25 μM) exaggerated the ACh-mediated inhibition of cAMP content, indicating that Ca _i ²⁺ affects cAMP production in SAN cells. In conclusion, muscarinic receptor stimulation inhibits the Ca _i ²⁺ transient via a cAMP-dependent signaling pathway. Inhibition of the Ca _i ²⁺ transient contributes to pacemaker slowing and inhibits Ca _i ²⁺ -stimulated cAMP production. Thus, we provide functional evidence for the contribution of the Ca _i ²⁺ transient to ACh-induced inhibition of pacemaker activity and cAMP content in rabbit SAN cells.     

Is pyruvate carboxylase involved in the renal tubular reabsorption of bicarbonate?

The co-existence of a hereditary defect of pyruvate carboxylase activity along with proximal renal tubular acidosis in several patients prompted the following theories:

1. Some of the bicarbonate which is normally reabsorbed from the glomerular filtrate is trapped in the mitochondria by pyruvate carboxylase in the conversion of pyruvate to oxaloacetate. The subsequent conversion of oxaloacetate to phosphoenol pyruvate releases CO₂ in the cytosol.
2. The trapping of HCO ₃ ^? by pyruvate (or other carboxylases) provides an important route for the recovery of filtered HCO ₃ ^? .
3. The process of trapping HCO ₃ ^? from the glomerular filtrate followed by release of CO₂ in the cytosol contributes to the apparently high RQ of kidney, since the CO₂ does not originate from a metabolic fuel.
4. Lactate and possibly other fuels are actively taken up by the kidney and are used as energy sources. Diversion of lactate for gluconeogenesis may contribute to the ‘excess substrate uptake’ phenomenon.
5. It is possible that some of the glucose which is synthesized in the cortex is used for glycolysis in the medulla. Conversely, lactate produced in the medulla may be available to the cortex for bicarbonate trapping and thus for gluconeogenesis.

     

Role of leptin as antioxidant in obstructive sleep apnea: an in vitro study using electron paramagnetic resonance method

Introduction

As in obstructive sleep apnea (OSA), the chronic cycles of hypoxia and reoxygenation are thought to be conducive of oxidative stress (OS) with generation of reactive oxygen species, identifying effective mechanisms of protection against oxidant-mediated tissue damage becomes of outmost importance. Leptin’s role had been recently extended into that of participant to OS; while its exact role in this process is yet to be defined, elevated leptin levels correlate significantly with several indices of OSA disease severity such as nocturnal hypoxemia, possibly acting as a counteractive mechanism against the chronic intermittent hypoxia-related OS and serving as a marker of future risk of atherosclerotic disease. We therefore investigated leptin’s antioxidant mechanism on superoxide (O ₂ ^?? ) anions using spectrophotometry and electron paramagnetic resonance (EPR).

Methods

The O ₂ ^?? was generated by oxidation of xanthine (XAN) by xanthine oxidase (XO) in the presence of spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide with various concentrations of leptin (0.001, 0.01, 0.1, and 1 mg/ml) and without leptin. Signal intensity between 3,440 and 3,540 G was expressed as standard means ± SD. The activity of leptin on XO was determined by monitoring the conversion of XAN to uric acid at 293 nm using a Beckman DU 800 UV–visible spectrophotometer.

Results

Leptin added to aqueous solutions at 0.1 and 1 mg/ml concentrations was associated with a statistically significant decrease in the EPR signal due to leptin’s direct scavenging activity towards the O ₂ ^?? .

Conclusion

Leptin is an antioxidant agent of possible use as a marker of OS and future risk of atherosclerotic disease in OSA.     

Buffering characteristics of eel blood at the extreme conditions in the swimbladder

CO₂ binding in whole blood and true plasma of the eel was estimated by measuring CO₂ content and pH in blood aliquots equilibrated with various P_CO2 values over a wide range expected to occur in the swimbladder. Bicarbonate concentration, [HCO₃⁻], was calculated using the CO₂ solubility coefficient, which was measured to average 50 μmol·L⁻¹·Torr⁻¹ (20°C). Buffer lines of non-bicarbonate buffers were obtained in plots of [HCPO₃⁻] against pH, and non-bicarbonate buffer values, β_NB, were obtained by curve. fitting.In the pH range 6.6–8.2, the buffer line for oxygenated whole blood was sigmoid, while that for deoxygenated blood increased its slope monotonously with increasing pH. The β_NB for oxygenated blood displayed a maximum of about 8.6 mmol·L⁻¹pH⁻¹ at pH = 7.4 and dropped down to below 1 mmol·L⁻¹·pH⁻¹ at higher and lower pH. Similar shapes of the buffer lines were obtaned in true plasma; the [HCO₃⁻] levels and β_NB values were, however, somewhat higher than in whole blood.These data are useful fo assess the back-diffusion of CO₂ and HCO₃⁻ in the rete mirabile of the fish swimbladder and to estimate the effects CO₂ back-diffusion exerts on the counter-current enhancement of O₂ in the rete.     

Evaluation of usefulness of pleural fluid adenosine deaminase in diagnosing tuberculous pleural effusion from empyema

ObjectiveTo evaluate the utility of adenosine deaminase activity in the pleural fluid for the diagnosis of tuberculous pleural effusion from empyema of non-tubercular origin.MethodA retrospective analysis of data was performed on patients who were diagnosed to have tuberculous pleural effusion and empyema of non tubercular origin. Among 46 patients at Kasturba Hospital, Manipal University, Manipal, Karnataka, India, from November 2012 to February 2013 who underwent pleural fluid adenosine deaminase estimation, 25 patients with tuberculous pleural effusion and 21 patients with empyema were diagnosed respectively. Adenosine deaminase in pleural fluid is estimated using colorimetric, Galanti and Guisti method.ResultsPleural fluid Adenosine Deaminase levels among tuberculous pleural effusion(109.38±53.83), empyema (141.20±71.69) with P=0.27.ConclusionPleural fluid adenosine deaminase alone cannot be used as a marker for the diagnosis of tuberculous pleural effusion.     

Pleural Tissue Hyaluronan Produced by Postmortem Ventilation in Rabbits

We developed a method that used Alcian blue bound to hyaluronan to measure pleural hyaluronan in rabbits postmortem. Rabbits were killed, then ventilated with 21% O₂–5% CO₂–74% N₂ for 3 h. The pleural liquid was removed by suction and 5 ml Alcian blue stock solution (0.33 mg/ml, 3.3 pH) was injected into each chest cavity. After 10 min, the Alcian blue solution was removed and the unbound Alcian blue solution (supernatant) separated by centrifugation and filtration. The supernatant transmissibility (T) was measured spectrophotometrically at 613 nm. Supernatant Alcian blue concentration (Cab) was obtained from a calibration curve of T versus dilutions of stock solution Cab. Alcian blue bound to pleural tissue hyaluronan was obtained by subtracting supernatant Cab from stock solution Cab. Pleural tissue hyaluronan was obtained from a calibration curve of hyaluronan versus Alcian blue bound to hyaluronan. Compared with control rabbits, pleural tissue hyaluronan (0.21 ± 0.04 mg/kg) increased twofold, whereas pleural liquid volume decreased by 30% after 3 h of ventilation. Pleural effusions present 3 h postmortem without ventilation did not change pleural tissue hyaluronan from control values. Thus ventilation-induced pleural liquid shear stress, not increased filtration, was the stimulus for the increased hyaluronan produced from pleural mesothelial cells. Accepted for publication: 16 September 1999     

Pulmonary Microvascular Pressure Profile During Development of Hydrostatic Edema

Objective: To measure, in intact closed chest, the pressure in the pulmonary microvasculature during transition to mild interstitial edema. Methods: In anesthetized spontaneously breathing rabbits, the pulmonary artery and left atrium were cannulated. Pleural windows were prepared to view the superficial pulmonary microvascular network through the intact parietal pleura. After intravenous infusion of 96.4 ± 12.3 ml of saline at a rate of 0.5 ml/kg h, the hydraulic pressure in the pulmonary microvessels (15–240 μm in diameter) were measured using glass pipettes driven through the pleural window and connected to a servonull system. Results: After saline, plasma protein concentration decreased from 6 ± 1 to 4.8 ± 0.5 g/dl; pulmonary arterial and left atrial pressures averaged 22.3 ± 6.4 and 2.3 ± 2 cm H₂O in control and 23.1 ± 4.2 and 4.2 ± 2 cm H₂O after infusion. After saline loading, 16.4% of total pressure drop occurred from pulmonary artery to 80-μm arterioles, 60.3% in 30–80-μm arterioles, 6.9% from 30-μm arterioles to 30-μm venules and 16.4% in the downstream segment. Conclusions: Mild interstitial edema induced, with respect to control, constriction of small arterioles and capillary recruitment to maintain a low capillary pressure. Hence, in initial edema, pulmonary circulation prevents further fluid filtration, acting like an intrinsic safety factor to delay development of severe edema.     

A novel biomarker in the diagnosis of parapneumonic effusion: neutrophil gelatinase-associated lipocalin

Background

The protein neutrophil gelatinase-associated lipocalin (NGAL) is a mediator synthesized and released by neutrophils. Its physiological function is as yet unclear. Levels in blood increase in several inflammatory diseases. High serum values indicate poor prognosis for several diseases. Pleural effusion may appear as the result of various pathologies. The most common cause is heart failure (HF). Other common causes include parapneumonic (PPE) and malignant (MPE) pleural effusions, and pulmonary embolism. Tubercular effusion (TE) is commonly encountered in Turkey and similar developing countries. The purpose of this study was to investigate the effectiveness of NGAL, a current inflammation marker, in discriminating between different etiological diseases that cause pleural effusion.

Methods

The study was performed at the Recep Tayyip Erdo?an University Faculty of Medicine Chest Diseases Clinic. One hundred patients were included in the study, 25 with parapneumonic effusion, 25 with heart failure-related effusion, 25 with tubercular effusion and 25 with cancer-related effusion. NGAL was measured in patients’ serum and pleural fluids.

Results

Serum NGAL levels in PPE (171?±?56 ng/ml) were significantly higher (p?<?0.001) than those in HF (86?±?31 ng/ml), CA (103?±?42 ng/ml) and TE (63?±?19 ng/ml). Pleural NGAL levels were also significantly higher in PPE compared to HF, MPE and TE (p?<?0.001). Serum NGAL levels exhibited a positive correlation with white blood cell (WBC), neutrophil, C-reactive protein (CRP), sedimentation, serum LDH, creatinine, pleural leukocyte and pleural neutrophil numbers. The most significant correlation was between NGAL level and WBC (p?<?0.001, r?=?0.579). Both serum and pleural NGAL levels are highly effective in differentiating patients with PPE from those without PPE (AUC: 0.910 and 0.790, respectively).

Conclusions

NGAL can be used in the diagnosis of diseases with an acute inflammatory course. Serum and pleural NGAL levels can differentiate PPE from other diseases causing pleural fluid with high sensitivity and specificity.

     

Effects of urinary ouabain-like factors and vanadium diascorbate on calcium mobilization in porcine inner medullary collecting duct cells: Comparison with the effects of ouabain and vasopressin

It is speculated that ouabain-like factors (OLF) play a role in the pathogenesis of volume-dependent hypertension. In previous studies we isolated a more polar OLF-1 and a more apolar OLF-2 from the urine of healthy subjects after 5 days on a high sodium intake (>400 mmol/day) by gel chromatography (Sephadex G-25 and G-10) and reverse-phase HPLC. We subsequently identified the chemical structure of OLF-2 as vanadium (V^IV ) diascorbate. OLF-1, OLF-2, and vanadium diascorbate inhibited dose-dependently porcine Na-K-ATPase in vitro. Because the inner medullary collecting duct (IMCD) plays a crucial role in the long-term regulation of body fluid volume, in the present study we investigated the effects of urinary OLF-1 and OLF-2, and of vanadium diascorbate in comparison to ouabain and vasopressin (AVP) on calcium mobilization, ie, on free calcium concentration [Ca²⁺]_i, in cultured porcine IMCD cells. [Ca²⁺]_i was determined by the fura-2 method in IMCD cells isolated by hypotonic treatment and density gradient centrifugation from fresh porcine kidneys. Assuming an approximate molecular weight (MW) of 400 for OLF-1 and OLF-2, OLF-1 (10⁻⁴ mol/L) produced a slow increase in [Ca²⁺]_i from 39 ± 10 to 169 ± 21 nmol/L (n = 7 ) after 4 min. Similarly, OLF-2 (10⁻⁴ mol/L) resulted in an increase in [Ca²⁺]_i from 74 ± 20 to 216 ± 52 nmol/L (n = 7) after 4 min. Vanadium diascorbate (MW 403) dose-dependently increased [Ca²⁺]_i . At a concentration of 10⁻⁶ mol/L it increased [Ca²⁺]_i from 46 ± 5 to 149 ± 9 nmol/L (n = 5) after 4 min. A similar slow increase in [Ca²⁺]_i was found with ouabain (10⁻⁶ mol/L), which increased [Ca²⁺]_i from 61 ± 22 to 180 ± 29 nmol/L (n = 5) after 4 min in contrast to AVP (10⁻⁷ mol/L), which rapidly increased [Ca²⁺]_i from 48 ± 10 to 299 ± 32 nmol/L (n = 4) within 30 sec. Thus, OLF-1, OLF-2, and Vanadium diascorbate, the active component of OLF-2, reveal similar effects as ouabain on IMCD cells, ie, they produce a slow increase in [Ca²⁺]_i as expected from inhibition of Na-K-ATPase. The physiologic or pathologic roles of these and additional OLF in body fluid and blood pressure regulation and in hypertension have yet to be evaluated.     

Central, renal and adrenal effects of lithium in man.

The effect of lithium on thirst and plasma vasopressin concentration was tested in seven subjects with affective psychiatric disorders. Mean ad libitum fluid intake was liberal but no different before (3,293 ml/day) and three to four weeks after treatment with lithium (3,443 ml/day). After fluid deprivation, plasma vasopressin was 1.5 ± 0.39 pg/ml before and 3.72 ± 0.55 pg/ml after treatment with lithium (p < 0.02) as plasma osmolalities and body weights were comparable. Urinary osmolalities were no different (735 versus 759 mOsm/kg) and did riot increase with exogenous vasopressin. With a water load, plasma vasopressin decreased 1.58 to 0.79 pg/ml (p < 0.05) before and from 2.68 to 0.91 pg/ml (p < 0.025) after treatment with lithium. The water load excreted in 4 hours was less during lithium therapy (66 versus 85 per cent, p < 0.05). Lithium therapy had no effect on plasma renin activity (PRA) or aldosterone. On standing, PRA increased from 2.27 to 5.28 ng/ml/hour (p < 0.05) before and from 2.19 to 7.59 ng/ ml/hour (p < 0.05) after lithium therapy. At the same time plasma aldosterone increased from 121 to 365 pg/ml (p < 0.05) before and from 76 to 436 pg/ml (p < 0.05) after treatment with lithium. Lithium had no effect on indices of proximal tubular function (HCO₃^?, HPO₄⁼, glucose, amino acid and uric acid excretion). A lower titratable acid excretion (21 ± 5 versus 32 ± 4 μeq/min, p < 0.05) and higher urine pH (5.40 versus 5.02, p < 0.05) was observed after NH₄Cl ingestion during lithium therapy as compared to control. In conclusion, three to four weeks of lithium therapy neither stimulates thirst nor suppresses vasopressin release; some of the polyuria in patients with affective disorders may be due to their liberal fluid intakes. Lithium does not alter base line or standing PRA, aldosterone or proximal tubular function. Lithium does, however, induce an incomplete renal tubular acidosis.     

Stimulation of duodenal bicarbonate secretion by misoprostol

Graded doses of topical misoprostol were examined for effects on duodenal bicarbonate output in fasted conscious rats and compared with perfusions of the H₂-receptor blocker cimetidine. Chronic loops (2 cm) of the proximal duodenum were prepared in male Sprague-Dawley rats weighing 250 g. A thin perfusion tube was placed in the duodenal bulb, and a permanent cannula drained the distal end to the exterior. The stomach and remaining duodenum with biliary and pancreatic ducts were then reanastomosed. During tests, the loops were perfused with isotonic saline at 0.5 ml/min and samples of 15-min effluents analyzed for HCO₃^? using a modified back-titration method. Drugs and stimulants were added to the perfusion fluid. Misoprostol at 10^?10 M significantly (P<0.02) elevated the duodenal HCO₃^? from basal value (N=10), and concentrations of 10^?10 to 4×10^?4 M produced dose-dependent increases of bicarbonate secretion, with a peak output of 49±7 μmol/cm/hr. A peak response of the same magnitude, 42±4 μmol/cm/hr, is observed after 5-min exposure of the loops to 150 mm HCl. The response to a single dose of 10^?4 M misoprostol was sustained, similar to the prolonged stimulatory effects of 5-min acid exposure. Perfusions of the loop with graded natural PGE₂ result in similar increases of duodenal HCO₃^?, but the peak may be lower and the response to a single dose is less sustained. The H₂-receptor blocker cimetidine, perfused at concentrations of 10^?7 to 10^?4 M failed to affect duodenal, HCO₃^? output. Misoprostol was a potent stimulator of HCO₃^? output in the proximal duodenum of the conscious rat, and the response to a single dose was sustained. Peak outputs during misoprostol perfusion reached the same magnitude as observed after exposure of the loops to 150 mm HCl.     

In vivo measurements of blood flow and bone metabolism in osteoarthritis

With increasing age, there may be a decrease in femoral blood flow. In some patients, this may result in local ischaemia, which subsequently may lead to local degenerative changes. Consequently, bone blood flow may play an important role in the aetiology of osteoarthritis of the hip. Little is known about bone blood flow in the femoral head of patients with advanced hip osteoarthritis. The purpose of this study was to evaluate bone blood flow and metabolism in vivo in patients with osteoarthritis of the hip. Ten patients with symptomatic osteoarthritis of the hip were enrolled prospectively. Femoral bone blood flow and metabolism were measured using positron emission tomography together with H ₂ ¹⁵ O and [¹⁸F]fluoride, respectively. Blood flow was 0.054 ± 0.032 mL cm^?3 min^?1 and 0.041 ± 0.012 mL cm^?3 min^?1 in symptomatic and contralateral femoral heads, respectively (p = 0.435). The net flux of fluoride from plasma to bone mineral (K _i ) was significantly (p = 0.027) higher in the femoral head of the osteoarthritic hip (0.022 ± 0.012 mL cm^?3 min^?1) than in that of the contralateral hip (0.007 ± 0.005 mL cm^?3 min^?1). This study showed significant increase in bone metabolism in the proximal femur of patients with symptomatic osteoarthritis of the hip joint. There was no evidence of decreased blood flow.     

Tumour necrosis factor-alpha in comparison to adenosine deaminase in tuberculous pleuritis

BACKGROUND: Adenosine deaminase (ADA) is already used for the differential diagnosis of tuberculosis pleurisy. Tumour necrosis factor-alpha (TNF) is another marker which has been investigated for this purpose. OBJECTIVE: We evaluated the diagnostic value of pleural fluid and serum TNF concentrations in tuberculous pleuritis and compared them to ADA. METHODS: Sixty-two patients (24 tuberculous pleuritis, 38 non-tuberculous pleuritis) with exudative pleurisy were included. Serum and pleural fluid TNF concentrations were determined in all patients and ADA activity in 54 patients. Pleural fluid TNF concentrations and pleural fluid/serum TNF were compared to pleural fluid ADA activity and pleural fluid/serum ADA. RESULTS: When the tuberculous and non-tuberculous groups were compared, pleural fluid TNF concentrations (65.4 +/- 136.9 pg/ml vs. 54.5 +/- 144.2 pg/ml, respectively; p < 0.001), pleural fluid ADA activity (74.2 +/- 33.3 U/l vs. 23 +/- 16.3 U/l; p < 0.0001), pleural fluid/serum TNF (2.55 +/- 5.23 vs. 0.26 +/- 0.2; p < 0.001) and pleural fluid/serum ADA (4.58 +/- 8.14 vs. 1.15 +/- 0.7; p < 0.0001) were significantly higher in the tuberculous group. When cut-off points were assessed, 8 pg/ml and 40 U/l were found for pleural fluid TNF concentrations and pleural fluid ADA activity, respectively. Sensitivity, specificity, area under the curve were 87.5%, 76.3%, 0.772 for pleural fluid TNF concentrations and 90.9%, 89.5%, 0.952 for pleural fluid ADA activity, respectively; the difference between these areas under the curves was significant (p < 0.05). CONCLUSIONS: Pleural fluid TNF levels and pleural fluid/serum TNF were higher in tuberculous effusions than in other exudates, but their diagnostic value appears to be poorer than that of ADA.     

Cerebral blood flow and cerebrovascular reactivity at rest and during sub-maximal exercise: Effect of age and 12-week exercise training

Chronic reductions in cerebral blood flow (CBF) and cerebrovascular reactivity to CO₂ are risk factors for cerebrovascular disease. Higher aerobic fitness is associated with higher CBF at any age; however, whether CBF or reactivity can be elevated following an exercise training intervention in healthy individuals is unknown. The aim of this study was to assess the effect of exercise training on CBF and cerebrovascular reactivity at rest and during exercise in young and older individuals. Ten young (23?±?5 years; body mass index (BMI), 26?±?3 kg m^?2; $ {\mathop{V}\limits^{ \cdot }{_{\text{O2}}}}\max $ , 35?±?5 ml kg^?1 min^?1) and 10 older (63?±?5 years; BMI, 25?±?3.0 kg m^?2; $ {\mathop{V}\limits^{ \cdot }{_{\text{O2}}}}\max $ , 26?±?4 ml kg^-1 min^?1) previously sedentary individuals breathed 5 % CO₂ for 3 min at rest and during steady-state cycling exercise (30 and 70 % heart rate range (HRR)) prior to and following a 12-week aerobic exercise intervention. Effects of training on middle cerebral artery blood velocity (MCAv) at rest were unclear in both age groups. The absolute MCAv response to exercise was greater in the young (9 and 9 cm s^?1 (30 and 70 % HRR, respectively) vs. 5 and 4 cm s^?1 (older), P?<?0.05) and was similar following training. Cerebrovascular reactivity was elevated following the 12-week training at rest (2.87?±?0.76 vs. 2.54?±?1.12 cm s^?1 mm Hg^?1, P?=?0.01) and during exercise, irrespective of age. The finding of a training-induced elevation in cerebrovascular reactivity provides further support for exercise as a preventative tool in cerebrovascular and neurological disease with ageing.     

Changes in serum amylase, lipase and leukocyte elastase during diabetic ketoacidosis and poorly controlled diabetes

Diabetic ketoacidosis (DKA) is frequently associated with pancreatic enzyme abnormalities. In order to determine the main factors that lead to this increase, serum total amylase (TA), pancreatic amylase (PA), lipase (L) and leukocyte elastase (LE), an early predictor of acute pancreatitis, were measured in four groups of patients on admission. Group 1 consisted of 52 patients with DKA (age: 41.9 ± 19.2 years; blood glucose (Glc): 27.4 ± 11.5 mmol/L; pH: 7.20 ± 0.16; plasma bicarbonate: 10.5 ± 6.2 mmol/L; blood urea nitrogen (BUN): 0.60 ± 0.44 g/L; HbA_1C: 12.5% ± 2.8%). Group 2 consisted of 90 patients with poorly controlled non-ketotic diabetes (age: 53.4 ± 16.0; Glc: 14.3 ± 0.6; HCO₃ ⁻: 26.6 ± 3.2; BUN: 0.38 ± 0.20; HbA_1C: 11.3 ± 2.1). Group 3 consisted of 22 patients with well-controlled diabetes (age: 53.7 ± 12.8; Glc: 10.1 ± 5.2; HCO₃ ⁻: 27.4 ± 3.8; BUN: 0.36 ± 0.19; HbA_1C: 6.8 ± 0.8). Group 4 (controls) comprised 27 non-diabetic patients (age: 46.0 ± 15.0; Glc: 4.9 ± 0.5; HCO₃ ⁻: 28.4 ± 2.5; BUN: 0.30 ± 0.16; HbA_1C: 5.2 ± 0.7) (means ± SD). Increased enzyme activities were more frequent in group 1 (TA: 30.7; PA: 27.0; L: 36.5; LE: 73%) than in groups 2 (TA: 8.9; PA: 7.1; L: 8.9; LE: 45.5%), 3 (TA: 13.6; PA: 9.0; L: 18.1; LE: 31.8%) and 4 (TA: 7.0; PA: 3.0; L: 0.0; LE: 29.6%). Mean serum enzyme activities were significantly different in the 4 groups (ANOVA, P < 0.01) and were higher in group 1 than in groups 2, 3 and 4 (Student's t-test; group 1 vs 2 or 3 or 4: P < 0.001). In groups 1 + 2 + 3 + 4 (all patients), the four enzymes correlated with one another and also with Glc, BUN and HCO₃ ⁻ (P < 0.001). In group 1, TA correlated negatively with HCO₃ ⁻ (P < 0.001) and pH (P < 0.05); PA and L correlated positively with Glc and BUN (P < 0.01) and negatively with HCO₃ ⁻ (respectively, p < 0.01 and 0.05). PA correlated positively with pH (P < 0.01); LE correlated with Glc (P < 0.05) and BUN (P < 0.01). In conclusion, this study suggests that the serum levels of pancreatic enzymes increase with the degree of diabetic disequilibrium, and mainly correlate with metabolic factors such as hyperglycaemia, dehydration and acidosis. Increased pancreatic enzyme activities in patients with DKA, even in combination with abdominal pain, should not be diagnosed as acute pancreatitis; this could be important, particularly for younger clinicians. Received: 18 September 1998 / Accepted in revised form: 24 February 1999     

l-arginine and endogenous nitric oxide protect the gastric mucosa from endothelin-1-induced gastric ulcers in rats

We have reported that endothelin-1 induces gastric ulcer characterized by a potent long-lasting vasoconstriction of the regional microvasculature. Nitric oxide synthesized froml-arginine has been shown to regulated gastric mucosal blood flow, and inhibition of its synthesis has been shown to delay the healing of gastric ulcers. We examined the effect of exogenousl-arginine and the inhibition of nitric oxide synthesis on the development of endothelin-1-induced gastric ulcers. In rats anesthetized with urethane, a continuous intravenous infusion ofl-ord-arginine (10 mg·kg^?1·min^?1) was followed, 15 min later, by a submucosal injection of endothelin-1 (200 pmol/kg) in the anterior wall of the gastric body. In another group, rats were intravenously pretreated with N^ω-nitro-l-arginine-methyl ester (1–10mg/kg), a nitric oxide synthesis inhibitor, and then injected with endothelin-1 (40 pmol/kg). Twenty-four h later,l-arginine, but notd-arginine, had significantly reduced the extent and the severity of the endothelin-1-induced ulcer (mucosal wall damage, 18.11 ± 4.79% and 88.14 ±7.06%, respectively; mean ± SD,P<0.001), and the nitric oxide synthesis inhibitor (10mg/kg) had increased the endothelin-1-induced mucosal damage (ulcer length, 3.8 ± 1.2 mm and 1.1 ± 0.2 mm, respectively,P<0.01). Continuous gastric mucosal blood flow measurements showed thatl-arginine antagonized the endothelin-1-induced vasoconstriction.l-arginine protected the gastric mucosa from the ulcerogenic action of endothelin-1 and antagonized its vasoconstrictive action. The inhibition of endogenous nitric oxide potentiated the ulcerogenic effect of endothelin-1 on rat gastric mucosa.     

Oscillations in oxygen tension and insulin release of individual pancreatic ob/ob mouse islets

Aims/hypothesis. The role of beta-cell metabolism for generation of oscillatory insulin release was investigated by simultaneous measurements of oxygen tension (pO₂) and insulin release from individual islets of Langerhans.¶Methods. Individual islets isolated from the ob/ob-mice were perifused. Insulin in the perifusate was measured with a sensitive ELISA and pO₂ with a modified Clark-type electrode inserted into the islets.¶Results. In the presence of 3 mmol/l d-glucose, pO₂ was 102 ± 9 mmHg and oscillatory (0.26 ± 0.04 oscillations/min). Corresponding insulin measurements showed oscillatory release with similar periodicity (0.25 ± 0.02 oscillations/min). When the d-glucose concentration was increased to 11 mmol/l, pO₂ decreased by 30 % to 72 ± 10 mmHg with maintained frequency of the oscillations. Corresponding insulin secretory rate rose from 5 ± 2 to 131 ± 16 pmol · g^–1· s^–1 leaving the frequency of the insulin pulses unaffected. The magnitude of glucose-induced change in pO₂ varied between islets but was positively correlated to the amount of insulin released (r ² = 0.85). When 1 mmol/l tolbutamide was added to the perifusion medium containing 11 mmol/l glucose no change in average oscillatory pO₂ was observed despite a doubling in the secretory rate. When 8 mmol/l 3-oxymethyl glucose was added to perifusion medium containing 3 mmol/l d-glucose, neither pO₂ nor insulin release of the islets were changed. Temporal analysis of oscillations in pO₂ and insulin release revealed that maximum respiration correlated to maximum or close to maximum insulin release.¶Conclusion/interpretation. The temporal relation between oscillations in pO₂ and insulin release supports a role for metabolic oscillations in the generation of pulsatile insulin release. [Diabetologia (2000) 43: 1313–1318]     

Atrial natriuretic peptide in Type 2 diabetes mellitus: response to a physiological mixed meal and relationship to renal function

Relatively few data exist on atrial natriuretic peptide (ANP) characteristics in Type 2 diabetes mellitus (DM). Therefore, plasma immunoreactive ANP concentrations were measured before and for 4 h following the ingestion of a physiological mixed meal in 8 newly diagnosed, normotensive, normoalbuminuric, patients with Type 2 DM and 6 normotensive, non-diabetic controls. In patients with Type 2 DM, basal plasma ANP concentrations were 4.0 ± 2.0 and not significantly changed following ingestion of the meal, with peak levels of 4.9 ± 2.8 pmol l⁻¹. Non-diabetic controls had higher basal plasma ANP concentrations, 8.7 ± 3.4 pmol l⁻¹ (p < 0.05), significantly increasing to a peak of 11.9 ± 6.3 pmol l⁻¹ at 30 min post meal. Extracellular fluid volume (ECV) was not different between diabetic patients and controls (15877 ± 2679 vs 13668 ± 1792 ml³). Glomerular filtration rate (GFR) (isotopic clearance corrected for body surface area) was elevated in diabetic patients (mean ± SD) 130 ± 39 vs 98 ± 10 ml min⁻¹, p < 0.05). For the DM subjects, basal ANP levels were negatively correlated with GFR (r_s − 0.74, p < 0.05) and effective renal plasma flow (ERPF) (r_s − 0.8, p < 0.05). We conclude that patients with Type 2 DM demonstrate reduced basal plasma ANP concentrations which are inversely correlated to renal function. In contrast to non-diabetic controls, ANP in Type 2 DM does not rise in response to feeding. © 1998 John Wiley & Sons, Ltd.     

Hemodynamic and functional consequences of intravascular platelet aggregation in skeletal muscle

The effect of ADP-induced platelet aggregation upon blood flow, O₂-consumption, and performance was investigated in isolated and autoperfused working canine gastrocnemius muscles. During steady state work a mean blood flow of 87.1 ± 33.7 ml × min^?1 × 100 g^?1 and a mean O₂-consumption of 7.89 ± 4.03 ml × min^?1 × 100 g^?1 was found. The average amplitude of muscle contraction was 10.4 ± 4.1 mm. Changes of these parameters due to the ADP-infusion were found to depend upon the number of platelets which remained in the muscles during infusion. On the other hand this so-called platelet loss was significantly correlated with the arterial platelet count. In animals with a low arterial platelet count (below 10⁴/mm³) platelet loss did not exceed 2 × 10⁹ cells × min^?1 × 100 g^?1. Blood flow as well as venous O₂-saturation increased during ADP-infusion. In an experiment with a high platelet loss (14.3 × 10⁹ cells × min^?1 × 100 g^?1) according to a platelet count of 390 × 10³/mm³ muscle contraction and O₂-consumption were reduced to approximately 30% of their preinfusion values. These changes, however, only were present during the time of ADP-infusion. After its termination the muscles started to recover immediately reaching the control values within 5 min.