Comparison of the activation of the Ca2+ release-activated Ca2+ current I CRAC to InsP3 in Jurkat T-lymphocytes, pulmonary artery endothelia and RBL-1 cells

In many electrically non-excitable cells, Ca2+ entry is mediated predominantly by the store-operated Ca2+ influx pathway. The best-characterised store-operated Ca2+ current is the Ca2+ release-activated Ca2+ current (ICRAC). It is generally believed that high concentrations of intracellular Ca2+ buffer are required to measure ICRAC, due to Ca2+-dependent inactivation of the channels. Recently, we have recorded robust ICRAC in rat basophilic leukaemia (RBL-1) cells at physiological levels of Ca2+ buffering when stores were depleted by inhibition of the sarcoplasmic/ endoplasmic reticulum Ca2+-activated adenosine triphosphatase (SERCA) pumps. However, the second messenger inositol 1,4,5-trisphosphate (InsP3) was not able to evoke the current under such conditions, despite inducing substantial Ca2+ release. We have therefore suggested that a threshold exists within the Ca2+ stores which has to be overcome for macroscopic ICRAC to activate. To establish whether this is a specific feature of ICRAC in RBL-1 cells or whether it is a more general phenomenon, we investigated whether a threshold is also seen in other cell-types used to study store-operated Ca2+ entry. In Jurkat-T lymphocytes, ICRAC is activated weakly by InsP3 in the presence of low concentrations of Ca2+ buffer, whereas the current is large when SERCA pumps are blocked simultaneously, as in RBL-1 cells. Although the electrophysiological properties of ICRAC in the Jurkat cell are very similar to those of RBL-1 cells, the Na+ conductance in the absence of external divalent cations is quite different. Unexpectedly, we failed consistently to record any store-operated Ca2+ current in macrovascular pulmonary artery endothelia whereas robust ICRAC was seen under the same conditions in RBL-1 cells. Our results show that ICRAC has a similar profile of activation in the presence of physiological levels of Ca2+ buffering for Jurkat T-lymphocytes and RBL-1 cells, indicating that the threshold mechanism may be a general feature of ICRAC activation. Because ICRAC in pulmonary artery endothelia is, at best, very small, additional Ca2+ influx pathways may also contribute to agonist-induced Ca2+ entry.     

Caffeine inhibits cytoplasmic Ca2+ oscillations induced by carbachol and guanosine 5′-O-(3-thiotriphosphate) in hyperpolarized pancreatic ß-cells

The effects of caffeine on cytoplasmic Ca²⁺ oscillations induced by carbachol and guanosine 5-O-(3-thiotriphosphate) (GTP--S) were studied in individual mouse pancreatic ß-cells clamped at a hyperpolarized potential. Addition of 10 mM caffeine did not affect the cytoplasmic Ca²⁺ concentration ([Ca²⁺]₁) in ß-cells exposed to 20 mM glucose and hyperpolarized with diazoxide. Under similar conditions 100 M carbachol induced a typical response with a marked [Ca²⁺]_i peak followed by a lower sustained elevation. Irrespective of whether 10 mM caffeine was present, there were [Ca²⁺]_i transients with frequencies of 1–5/min superimposed on the sustained phase in 50–60% of the cells. In previously non-exposed cells the introduction of 10 mM caffeine caused temporary lowering of the sustained phase with disappearance of the transients. Subsequent omission of caffeine in the continued presence of carbachol caused a marked [Ca²⁺]_i peak followed by reappearance of the [Ca²⁺]_i, transients. However, in cells oscillating in the presence of caffeine its omission caused disappearance of the transients. In this case reintroduction of caffeine restored the transients.In cells kept at –70 mV by a patch pipette containing 100 M GTP--S and 3 mM Mg-ATP there were [Ca²⁺]_i transients with frequencies of 0.5–2.5/min. These transients were sufficiently pronounced to activate repetitively a K⁺ current. Addition of 10 mM caffeine caused disappearance of the [Ca²⁺]_i transients or reduction of their amplitudes and frequencies.The results indicate that caffeine does not activate Ca²⁺-induced Ca²⁺ release in hyperpolarized ß-cells but inhibits the Ca²⁺-mobilizing effect of inositol 1,4,5-trisphosphate. Correspondence to: E. Gylfe at the above address     

Serum C-reactive protein in elderly men and women: association with mortality, morbidity and various biochemical values

OBJECTIVE: The primary aim of this study was to define the distribution and the prognostic value of serum C-reactive protein (s-CRP) measured by a high-sensitivity method in elderly subjects of both genders with special reference to the distribution below 10 mg/l. As a secondary aim, a possible gender difference of s-CRP was examined. MATERIAL AND METHODS: Baseline s-CRP was described in a population-based sample of opposite-sex, twin-pairs (197 F, 189 M available for blood-sampling) aged 71-80 years (mean age 74.5 years), considering mortality through the next 4 years, morbidity (myocardial infarction, angina pectoris, congestive heart failure, arterial hypertension, venous thromboembolism, stroke, diabetes, gout, psoriasis, rheumatoid arthritis) before and after blood sampling, biochemical values (serum levels of urate, urea, ApoA1, ApoB, folate, FSH, LH, oestradiol, progesterone, testosterone, cortisol) and anthropometric measurements (body mass index (BMI), circumference of waist, buttocks and hips). RESULTS: The level of s-CRP did not deviate substantially from what has been reported for younger subjects. Higher values indicated an increased risk of cardiovascular morbidity and diabetes in women but not in men. The s-CRP level was associated with serum levels of urate, progesterone, folate, ApoA1, ApoB and the quotient ApoB/ApoA1 as well as with BMI and waist circumference. CONCLUSIONS: For the 71-80 years age group, s-CRP below the 80th percentile (4.3 mg/l) seems to have prognostic capacity mainly in women. The highest association with mortality as well as with cardiovascular diseases, diabetes and rheumatoid arthritis is found for s-CRP above 10 mg/l, which is the arbitrary lower level for the earlier routine low-sensitivity s-CRP methods. The association of s-CRP with serum urate, folate and the ApoB/ApoA1 quotient should be considered.     

Urine Dipstick of Sputum for the Rapid Diagnosis of Community Acquired Pneumonia

Introduction

Community acquired pneumonia (CAP) is responsible for an important part of treatment costs across the world. Even though posterior-anterior lung radiography (PALG) and direct sputum smear microscopy are required or routine diagnoses. The purpose of this study is to determine the diagnostic value of the bedside urine strip tests in CAP.

Measurement of lumefantrine and its metabolite in plasma by high performance liquid chromatography with ultraviolet detection

Artemether-lumefantrine (ARM-LUM) has in recent years become the first-line treatment for uncomplicated malaria in many Sub-Saharan African countries. Vigorous monitoring of the therapeutic efficacy of this treatment is needed. This requires high-quality studies following standard protocols; ideally, such studies should incorporate measurement of drug levels in the study patients to exclude the possibility that insufficient drug levels explain an observed treatment failure. Several methods for measuring lumefantrine (LUM) in plasma by HPLC are available; however, several of these methods have some limitations in terms of high costs and limited feasibility arising from large required sample volumes and demanding sample preparation. Therefore, we set out to develop a simpler reversed phase high performance liquid chromatography (RP-HPLC) method based on UV detection for simultaneous measurement of LUM and its major metabolite the desbutyl LUM (DL) in plasma. Halofantrine was used as an internal standard. Liquid–liquid extraction of samples was carried out using hexane–ethyl acetate (70:30, v/v). Chromatographic separation was carried out on a Synergi Polar-RP column (250 mm × 300 mm, particle size 4 μm). The mobile phase consisted of acetonitrile–0.1 M ammonium acetate buffer adjusted to pH 4.9 (85:15%, v/v). Absorbance of the compounds was monitored at 335 nm using a reference wavelength of 360 nm. Absolute extraction recovery for LUM and DL were 88% and 90%, respectively. Inter- and intraday coefficients of variation for LUM and DL were ≤10%. The lower limits of quantification for LUM and DL were 12.5 and 6.5 ng/ml, respectively. After validation, the methodology was transferred to a local laboratory in Tanga Tanzania and samples from a small subset of malaria patients were analysed for LUM. The method appears to be applicable in settings with limited facilities.     

Selective effect of some somatostatin analogs on glucagon as opposed to insulin release in rats in vivo

Cyclic somatostatin, at a dose of 700 but not 70 ng/kg/min, inhibited arginine-induced insulin and glucagon release as well as glucose stimulated insulin release in rats in vivo. Three somatostatin (S-S) analogs (D-Cys14-S-S, D-Trp8-D-Cys14-S-S and Ala2-D-Trp8-D-Cys14-S-S), at a dose of 70 ng/kg/min, suppressed arginine-induced glucagon but not insulin release. At the same dose, the first two of these analogs had no effect on glucose-induced insulin release, while the third one. Ala2-D-Trp8-D-Cys14-somatostatin, enhanced insulin release induced by glucose. A fourth analog, D-Trp8-somatostatin, was more potent than somatostatin with regard to arginine stimulated insulin and glucagon release, and equipotent with somatostatin with respect to glucose stimulated insulin release. These studies show, firstly, that the inhibitory effect of somatostatin analogs on arginine induced insulin release may be different from that when glucose is used as a stimulant and, secondly, that Ala2-D-Trp8-D-Cys14-somatostatin inhibits arginine-induced glucagon release while enhancing insulin release on glucose stimulation.     

Patient management of long-term continuous subcutaneous insulin infusion

AIM: This paper reports a study of patients' current practice with continuous subcutaneous insulin infusions, particularly with respect to the management of the pump. BACKGROUND: Successful implementation of continuous subcutaneous insulin infusion requires a motivated patient with a range of technical skills and self-management capabilities. The therapy should be prescribed, implemented and monitored by a skilled professional team familiar with it and capable of supporting the patient. METHODS: A questionnaire was mailed to 102 continuous subcutaneous insulin infusion treated patients at a Swedish university hospital with experience of pump treatment for at least 6 months. RESULTS: The questionnaire was answered by 88% of the patients, 53 women and 37 men, aged 22-71 years with a duration of continuous subcutaneous insulin infusion use of between 7 months and 19 years. The changing interval for soft infusion set ranged from 2.0 to 10.0 days (mean 4.8) and for metal needles from 1.5 to 7.5 days (mean 3.8), P = 0.001. Catheter occlusions were significantly more often reported in patients with presence of bleeding at the infusion site (P = 0.011) and among those using insulin lispro (P = 0.032). CONCLUSIONS: Patients having long-term continuous subcutaneous insulin infusion should be carefully audited with respect to the management of the insulin pump and its accessories. In patients who frequently experience problems, shorter intervals between changes of infusion sets are strongly advocated and type of insulin preparation may be of importance in some cases.     

Serum 2 -microglobulin in various disorders

In 216 patients admitted to hospital for various disorders serum β₂-microglobulin was determined by a radioimmunoassay. Only subjects with a serum creatinine value in the lower half of the normal range were studied as the level of serum β₂-microglobulin is known to increase when there is an impairment of the renal function. Most patients had normal serum levels but high values of serum β₂-microglobulin were relatively often found in patients with malignant disease. Elevated values were also found in a few subjects with inflammatory disorders thought to be connected with a pronounced or abnormal immune response.