Lack of relationship between serum and gallbladder bile calcium in patients with gallstone disease

BACKGROUND: Recent studies suggest that alternation in serum calcium influences the level of gallbladder bile ionized calcium (Ca2+). Theoretically, this could increase the risk of calcium precipitation in the gallbladder. METHODS: We therefore measured serum and gallbladder bile minerals in patients with gallstones (n = 27) and without (n = 10, controls). The serum samples were taken just prior to induction of anaesthesia and gallbladder bile was aspirated before any manipulation of the gallbladder. RESULTS: The active molality of Ca2+ in gallbladder bile was not statistically significant different between cases and controls (0.44 +/- 0.16 vs. 0.40 +/- 0.10 mmol/kg), whereas pH was significantly lower (6.94 +/- 0.31 vs. 7.36 +/- 0.28, p < 0.0001) and cholesterol higher (4.37 +/- 2.70 vs. 1.79 +/- 1.33 mmol/l; p < 0.01) in gallbladder bile obtained from cases. Serum Ca2+ at actual pH, magnesium and phosphate were significantly higher among cases than in controls. Gallbladder bile active molality of Ca2+ was significantly correlated with bile total calcium in both groups (r = 0.72; p < 0.001 and r = 0.91; p < 0.001, respectively). In controls only, we observed a positive relationship between serum Ca2+ at actual pH and the active molality of Ca2+ in bile (r = 0.61; p < 0.05). CONCLUSION: Our study demonstrates that Ca2+ in gallbladder bile does not differ between cases and controls. The lack of correlation between serum and gallbladder bile constituents in cases compared to controls suggests that changes in calcium equilibration between bile and serum in patients with gallstone disease might be of importance for the formation of gallstones.     

Electrolytes and pH changes in pre-eclamptic rats

BACKGROUND: Intracellular free calcium [Ca2+]i and magnesium [Mg2+]i ions play major roles in the mechanism of vascular smooth muscle (VSM) contraction. Although essential hypertension and abnormal intracellular homeostasis of these ions have long been recognized as major icons in the pathogenesis of pre-eclampsia, the underlying mechanism(s) remain poorly understood. METHODS: Alterations of vascular smooth muscle and platelet intracellular cations [Ca2+]i, [Mg2+]i and [H+]i relative to plasma concentrations of these ions in nitric oxide synthase (NOS) blockade-induced models of pre-eclampsia have been evaluated in the present study. RESULTS: Pregnant rats injected with the NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) developed a significantly elevated arterial blood pressure, proteinuria and other clinical parameters characteristic of pre-eclampsia compared to age-matched pregnant and non-pregnant rat controls that received the L-NAME vehicle only. Plasma total calcium concentration was significantly lower in pre-eclamptic rat models compared to normal pregnant rats (10.29+/-0.08 vs 10.67+/-0.18 mg/dl, p<0.05). A significant increase in plasma calcium was observed in pregnant controls compared to non-pregnant rats (10.67+/-0.18 vs 10.14+/-0.09 mg/dl, p<0.01). Plasma Ca2+ levels in pre-eclamptic rats were consistently lower than those of pregnant controls (5.69+/-0.09 vs 5.98+/-0.06 mg/dl, p<0.05). Resting levels of [Ca2+]i was significantly higher in pre-eclamptic rats than in pregnant controls. (351+/-45.2 vs 196+/-23.2 nmol/l, p<0.01). Blood pH was significantly increased in pre-eclamptic rats as compared to pregnant controls (7.16+/-0.02 vs 7.05+/-0.03, p<0.05). There was no significant difference in plasma and intracellular magnesium concentrations between the three rat groups. CONCLUSIONS: These findings suggest that a significantly decreased plasma level of Ca2+ coupled with a concomitant increase in VSM [Ca2+]i concentrations and an altered blood pH are associated with pre-eclampsia in the pregnant rat. Routine monitoring of serum pH, Ca2+ and Mg2+ especially in the late third trimester, may have potential in the early detection of patients at risk for pre-eclampsia, and monitoring the progress of diverse therapeutic regimens during clinical management.     

Glycolytic inhibition: effects on diastolic relaxation and intracellular calcium handling in hypertrophied rat ventricular myocytes.

We tested the hypothesis that glycolytic inhibition by 2-deoxyglucose causes greater impairment of diastolic relaxation and intracellular calcium handling in well-oxygenated hypertrophied adult rat myocytes compared with control myocytes. We simultaneously measured cell motion and intracellular free calcium concentration ([Ca2+]i) with indo-1 in isolated paced myocytes from aortic-banded rats and sham-operated rats. There was no difference in either the end-diastolic or peak-systolic [Ca2+]i between control and hypertrophied myocytes (97 +/- 18 vs. 105 +/- 15 nM, 467 +/- 92 vs. 556 +/- 67 nM, respectively). Myocytes were first superfused with oxygenated Hepes-buffered solution containing 1.2 mM CaCl2, 5.6 mM glucose, and 5 mM acetate, and paced at 3 Hz at 36 degrees C. Exposure to 20 mM 2-deoxyglucose as substitution of glucose for 15 min caused an upward shift of end-diastolic cell position in both control (n = 5) and hypertrophied myocytes (n = 10) (P < 0.001 vs. baseline), indicating an impaired extent of relaxation. Hypertrophied myocytes, however, showed a greater upward shift in end-diastolic cell position and slowing of relaxation compared with control myocytes (delta 144 +/- 28 vs. 55 +/- 15% of baseline diastolic position, P < 0.02). Exposure to 2-deoxyglucose increased end-diastolic [Ca2+]i in both groups (P < 0.001 vs. baseline), but there was no difference between hypertrophied and control myocytes (218 +/- 38 vs. 183 +/- 29 nM, respectively). The effects of 2-deoxyglucose were corroborated in isolated oxygenated perfused hearts in which glycolytic inhibition which caused severe elevation of isovolumic diastolic pressure and prolongation of relaxation in the hypertrophied hearts compared with controls. In summary, the inhibition of the glycolytic pathway impairs diastolic relaxation to a greater extent in hypertrophied myocytes than in control myocytes even in well-oxygenated conditions. The severe impairment of diastolic relaxation induced by 2-deoxyglucose in hypertrophied myocytes compared with control myocytes cannot be explained by greater diastolic Ca2+ overload, which implicates an increase in myofilament Ca(2+)-responsiveness as a possible mechanism.     

Dehydroepiandrosterone (DHEA) modulates the activity and the expression of lymphocyte subpopulations induced by cecal ligation and puncture

Dehydroepiandrosterone (DHEA) exerts a variety of positive effects on the immunologic alterations after trauma and sepsis. We therefore measured the therapeutic efficacy of DHEA after cecal ligation and puncture (CLP) on the expression of lymphocyte subpopulations and on the delayed type hypersensitivity (DTH) reaction. Male NMRI-mice were randomly assigned to four different treatment groups. Treatment consisted of DHEA or saline (S) administration after CLP or laparotomy only. Flow cytometry was performed (CD4+, CD8+, and CD56 lymphocytes) after 96 hours. DTH-reaction, activity and mortality rate were documented. The CLP-induced reduction in activity and survival (mortality: 34/40) was significantly (p < 0.03) less sustained in CLP-DHEA (mortality: 22/40). The DTH-ratio (before vs. after secondary challenge) was significantly lowered in CLP-S (1.01 +/- 0.15) compared to CLP-DHEA (1.35 +/- 0.1) after 48 hours (p < 0.01). CLP-DHEA (22.2 +/- 7.9%) was associated with a statistically significant less sustained increase of CD56+ cells (p < 0.01) compared with CLP-S (49.0 +/- 6.9%). DHEA-treatment after CLP was associated with less reduction in the CD8+ T-lymphocyte subsets (p < 0.01 vs. all other groups). DHEA treatment after CLP was associated with fewer alterations in the changes of CD8+ and CD56, cells, and the DTH reaction compared with animals submitted to CLP without any treatment. This difference was associated with improved outcome (reactivity, mortality). These results suggest a modulation at specific immune reactions by DHEA treatment.     

Low-dose intramuscular polymyxin B improves survival of septic rats

Polymyxin B (PLB) is a cationic antibiotic that also stoichiometrically neutralizes the lipid A moiety of endotoxin. We examined effects of a small dose of PLB on the mortality of rats with cecal ligation and puncture, on LPS-stimulated nitric oxide (NO) production, and on tumor necrosis factor alpha (TNF alpha) production by isolated rat Kupffer cells. MATERIALS AND METHODS: In vivo studies: Cecal ligation and puncture (CLP) was performed under anesthesia in 28 rats. One hour after CLP, either 600 U/kg of PLB or saline was administered intramuscularly every 6 h (PLB group: n = 12; control group: n = 16). Plasma endotoxin was measured at 3 and 24 h after the CLP by the Endospecy test. This was compared with survival. In vitro studies: Kupffer cells were isolated from the normal rat liver. The cells were incubated with LPS or LPS + PLB. After 24 h, NO and TNF alpha content were measured using the Griess and ELISA methods, respectively. RESULTS: Low dose PLB significantly decreased the endotoxin levels at both 3 and 24 h (5.5 +/- 2.1 pg/mL vs. 32.8 +/- 3.6 at 3 h; 26.1 +/- 6.1 vs. 49.1 +/- 5.6 at 24 h (p < .05) after CLP. PLB significantly improved survival of CLP rats (68.8% in the control group vs. 100% in the PLB treated group on 3 days after CLP, p < .001). PLB also attenuated NO and TNF alpha production from the Kupffer cells. CONCLUSION: Intramuscular PLB administered in low doses may improve the mortality of sepsis.     

Possible role of cytosolic free calcium concentrations in mediating insulin resistance of obesity and hyperinsulinemia.

Insulin- and glyburide-stimulated changes in cytosolic free calcium concentrations [( Ca2+]i) were studied in gluteal adipocytes obtained from six obese women (139 +/- 3% ideal body wt) and six healthy, normal weight age- and sex-matched controls. Biopsies were performed after an overnight fast and twice (at 3 and 6 h) during an insulin infusion (40 mU/m2 per min) (euglycemic clamp). In adipocytes obtained from normal subjects before insulin infusion, insulin (10 ng/ml) increased [Ca2+]i from 146 +/- 26 nM to 391 +/- 66 nM. Similar increases were evoked by 2 microM glyburide (329 +/- 41 nM). After 3 h of insulin infusion, basal [Ca2+]i rose to 234 +/- 21 nM, but the responses to insulin and glyburide were completely abolished. In vitro insulin-stimulated 2-deoxyglucose uptake was reduced by insulin and glucose infusion (25% stimulation before infusion, 5.4% at 3 h, and 0.85% at 6 h of infusion). In obese patients, basal adipocyte [Ca2+]i was increased (203 +/- 14 nM, P less than 0.05 vs. normals). The [Ca2+]i response demonstrated resistance to insulin (230 +/- 23 nM) and glyburide (249 +/- 19 nM) stimulation. Continuous insulin infusion increased basal [Ca2+]i (244 +/- 24 nM) and there was no response to either insulin or glyburide at 3 and 6 h of study. Rat adipocytes were preincubated with 1-10 mM glucose and 10 ng/ml insulin for 24 h. Measurements of 2-deoxyglucose uptake demonstrated insulin resistance in these cells. Under these experimental conditions, increased levels of [Ca2+]i that were no longer responsive to insulin were demonstrated. Verapamil in the preincubation medium prevented the development of insulin resistance.     

Calcium dependency and free calcium concentrations during insulin secretion in a hamster beta cell line.

Using a glucose-responsive beta cell line, we tested the hypothesis that the free cytosolic Ca2+ concentration ([Ca2+]i) is the primary signal that couples a stimulus to insulin secretion, and examined the involvement of the extracellular Ca2+ pool in this process. Glucose or depolarization of the beta cell with 40 mM K+ stimulated a monophasic release of insulin directly proportional to the extracellular Ca2+ concentration. 40 mM K+ increased 45Ca2+ uptake and increased [Ca2+]i, which was measured with quin 2, 4.7-fold, from 56 +/- 3 to 238 +/- 17 nM. With high glucose, 45Ca2+ uptake did not increase, and [Ca2+]i was unchanged or fell slightly. There was a striking correlation between inhibitory effects of verapamil, the Ca2+ channel blocker, on insulin secretion and the rise in [Ca2+]i evoked by K+. Higher concentrations of verapamil were required to inhibit glucose- than K+-stimulated insulin secretion (dose giving half-maximal effect of 1.4 X 10(-4) M vs. 6.0 X 10(-7) M). Incubation in Ca2+-free, 1 mM EGTA buffer for 30 min lowered [Ca2+]i to 14 +/- 2 nM, and inhibited acute insulin release to both secretagogues. If high glucose was present in the Ca2+-free period, reintroduction of 2.5 mM Ca2+ in high glucose restored insulin secretion only to the basal rate. However, if low glucose was present during the Ca2+-free period, high glucose and 2.5 mM Ca2+ triggered a full first-phase insulin response. These data suggest that high glucose generates a non-Ca2+ signal that turns over rapidly and provide direct evidence that K+ triggers insulin release by drawing extracellular Ca2+ into the beta cell through verapamil-sensitive Ca2+ channels. However, an increase [Ca2+]i is not the primary signal that evokes glucose-stimulated insulin release in this beta cell line.     

Blood pressure development of the spontaneously hypertensive rat after concurrent manipulations of dietary Ca2+ and Na+. Relation to intestinal Ca2+ fluxes.

The blood pressure of the spontaneously hypertensive rat (SHR) is influenced by the Ca2+ content of its diet. As the SHR's greater dependence on dietary calcium may reflect a defect in intestinal calcium absorption, we measured in vitro unidirectional Ca2+ flux (J) in the duodenum-jejunum (four segments each) of the SHR (n = 6) and the normotensive Wistar-Kyoto rat (WKY; n = 6) by a modified Ussing apparatus. Because of the known and postulated interactions between Ca2+ and Na+ in both intestinal and vascular tissue, we assessed in vivo the influence of a concurrent manipulation of Na+ intake (three levels: 0.25%, 0.45%, and 1.0%) on the blood pressure development of SHRs (n = 35) and WKYs (n = 35), between 6 and 20 wk of age, exposed to three levels of dietary calcium (0.1, 1.0, and 2%). Net calcium flux (Jnet) (mean +/- SEM) was significantly (P less than 0.01) lower in the SHR (-2.8 +/- 6.3 nmol/cm2 X h) than in the WKY (34.6 +/- 8.8 nmol/cm2 X h). The SHR's decreased Jnet resulted from a significantly (P less than 0.03) lower mucosa-to-serosa flux (Jm-s) in the SHR (41.0 +/- 5.6 nmol/cm2 X h) compared with the Jm-s of the WKY (70.1 +/- 9.1 nmol/cm2 X h). Serosa-to-mucosa flux for calcium did not differ between the SHR (43.8 +/- 6.6 nmol/cm2 X h) and the WKY (35.5 +/- 8.0 nmol/cm2 X h). The SHR's decreased (P less than 0.002) Jm-s was confirmed by additional measurements in SHRs and WKYs. Jm-s was 36.2 +/- 3.7 nmol/cm2 X h in the SHRs (n = 11) and 64.4 +/- 6.7 nmol/cm2 X h in the WKYs (n = 9). The provision of an increased dietary Ca2+ (2% by weight) and increased Na+ (1%) to the SHR prevented the emergence of hypertension (P less than 0.001) (mean +/- SEM systolic blood pressure at 20 wk of age; 135 +/- 5 mmHg for the 2% Ca2+, 1% Na+ SHR vs. 164 +/- 2 mmHg for the control diet SHR). Ca2+ (0.1%) and Na+ (0.25%) restriction accelerated the SHR's hypertension (192 +/- 2 mmHg) (P less than 0.001) and was associated with higher pressures in the WKY (146 +/- 4 mmHg in the restricted WKY vs. 134 +/- 4 mmHg in the control WKY). In a parallel group of 24 SHRs and 24 WKYs fed one of three diets (2% Ca2+/1% Na+; 1% Ca2+/0.45% Na+; or 0.1% Ca2+/0.25% Na+), the heart (P < 0.05) and kidney (P = 0.08) weight of the SHRs varied depending on the diet at 20 wk of age. Low Ca2+ and Na+ intake was associated with increased heart weight (1.6+/-0.9 g) compared with the normal diet for SHR (1.51+/-0.07 g). Increased Ca2+ and Na+ intake was associated with a significantly (P = 0.05) lower heart weight in the SHR (1.37+/-0.03 g) and in the WKY (1.35+/-0.06 g) compared with their normal diet controls. These findings show one mechanism for the SHR's depressor response to supplemental dietary Ca2+ and, in part, explain the sodium dependence of calcium's cardiovascular protective effect.     

The amiodarone derivative KB130015 [2-methyl-3-(3,5-diiodo-4-carboxymethoxybenzyl)benzofuran] induces an Na+-dependent increase of [Ca2+] in ventricular myocytes

KB130015 [KB; 2-methyl-3-(3,5-diiodo-4-carboxymethoxybenzyl)benzofuran] is a novel amiodarone derivative designed to retain the antiarrhythmic effects without the side effects. Unlike amiodarone, KB slows Na(+) current inactivation and could, via an increase in [Na(+)](i), potentially lead to Ca(2+) overload. Therefore, we studied the effects of KB on Na(+) and Ca(2+) handling in single pig ventricular myocytes using the whole-cell ruptured patch-clamp technique and K(5)fluo-3 as [Ca(2+)](i) indicator. KB at 10 microM did not prolong action potential duration but slightly increased the early plateau; spontaneous afterdepolarizations were not observed. The amplitude of the [Ca(2+)](i) transient was larger (434.9 +/- 37.2 versus 326.8 +/- 39.8 nM at baseline, n = 13, P < 0.05), and the time to peak [Ca(2+)](i) was prolonged. During voltage-clamp pulses, [Ca(2+)](i) transient peak was also larger (578.1 +/- 98.9 versus 346.4 +/- 52.6 nM at baseline, P < 0.05). Although L-type Ca(2+) current was reduced (by 21.9% at +10 mV, n = 9, P < 0.05), sarcoplasmic reticulum Ca(2+) content was significantly enhanced with KB. Forward Na(+)/Ca(2+) exchange was significantly decreased after KB application, but reverse mode of the Na(+)/Ca(2+) exchanger was significantly larger, suggesting an increase in [Na(+)](i) with KB. This was confirmed by a 2-fold increase of the [Na(+)]-dependent current generated by the Na/K-ATPase (from 0.17 +/- 0.02 to 0.38 +/- 0.06 pA/pF, P < 0.05). In conclusion, as predicted from the slowing of I(Na) inactivation, KB130015 leads to an increase in [Na(+)](i) and consequently in cellular Ca(2+) load. This effect is partially offset by a decrease in I(CaL) resulting in a mild inotropic effect without the signs of Ca(2+) overload and related arrhythmias usually associated with Na(+) channel openers.     

Age-dependent increase in arterial smooth muscle calcium in spontaneously hypertensive rats

Alterations in cellular calcium metabolism in essential hypertensive and in the SHR have been described. In the present study, particle-induced X-ray emission (PIXE) was used to get some information on the spatial distribution of Ca2+ in aortas of spontaneously hypertensive rats (SHR) and normotensive controls aged 1 week, 4 weeks, and 12 weeks. It was found that the Ca2+ content was not elevated in the aortic smooth muscle of SHR aged 1 week (n = 9) as compared to normotensive controls (n = 8) (186.8 +/- 89.9 micrograms Ca2+/g tissue vs 254.0 +/- 73.7 micrograms Ca2+/g tissue. The Ca2+ content was significantly raised in the aortic smooth muscle of SHR aged 4 weeks (n = 9) as compared to 4-weeks-old WKY rats (n = 12) (726.0 +/- 130.4 Ca2+/g tissue vs 440.3 +/- 214.4 Ca2+ micrograms/g tissue and in SHR aged 3 months (n = 15) as compared to WKY rats (n = 12), respectively (3317.0 +/- 734.0 micrograms Ca2+/g tissue vs. 1632.0 +/- 569.6 micrograms Ca2+/g tissue). The results confirm the age-related increase in the arterial Ca2+ content in normotensive rats and demonstrate additionally that this age-related rise in arterial Ca2+ content is accelerated in SHR.     

Intracellular acidification associated with changes in free cytosolic calcium. Evidence for Ca2+/H+ exchange via a plasma membrane Ca(2+)-ATPase in vascular smooth muscle cells.

The purpose of this study was to define the mechanism whereby agonists that increase free cytosolic calcium (Cai2+) affect intracellular pH (pHi) in smooth muscle. Rat aortic vascular smooth muscle cells grown on coverslips were loaded with BCECF/AM or fura-2/AM for continuous monitoring of pHi or Cai2+, respectively, in a HCO3-/CO2- containing medium. Recovery from rapid increases in Cai2+ produced by 1 microM angiotensin (Ang) II (delta Cai2+ -229 +/- 43 nM) or 1 microM ionomycin (delta Cai2+ -148 +/- 19 nM) was accompanied by a fall in pHi (delta pHi, -0.064 +/- 0.0085 P < 0.01, and -0.05 +/- 0.012 pH units, P < 0.01, respectively). Neither the fall in pHi nor the rise in Cai2+ elicited by Ang II was prevented by pretreatment with agents which block the action of this agonist on pHi via the stimulation of the Cl/HCo3 exchangers (DIDS, 50 microM) or the Na+/H+ antiporter (EIPA, 50 microM). In the presence of DIDS and EIPA, Ang II produced a fall in pHi (delta pHi, -0.050 +/- 0.014, P < 0.01) and a rise in Cai2+ (delta Ca2+ 252 +/- 157 nM, P < 0.01). That the change in pHi was secondary to changes in Cai2+ was inferred from the finding that, when the rise in Cai2+ elicited by Ang II was prevented by preincubation with a Ca2+ buffer, BAPTA (60 microM), the fall in pHi was abolished as well (delta pHi, 0.0014 +/- 0.0046). The pHi fall produced by Ang II and ionomycin was prevented by cadmium at a very low concentration (20 nM) which is known to inhibit plasma membrane Ca(2+)-ATPase activity (delta pHi -0.002 +/- 0.0006 and -0.0016 pH units, respectively). Cadmium also blunted Cai2+ recovery after Ang II and ionomycin. These findings suggest that the fall in pHi produced by these agents is due to H+ entry coupled to Ca2+ extrusion via the plasma membrane Ca(2+)-ATPase. Our results indicate that agonists that increase Cai2+ cause intracellular acidification as a result of Ca2+/H+ exchange across the plasma membrane. This process appears to be mediated by a plasma membrane Ca(2+)-ATPase which, in the process of extruding Ca2+ from the cell, brings in [H+] and thus acidifies the cell.     

Functional impairment of renal afferent arteriolar voltage-gated calcium channels in rats with diabetes mellitus.

Experiments were performed to test the hypothesis that diabetes mellitus is associated with impaired afferent arteriolar responsiveness to opening of voltage-gated calcium channels. Diabetes was induced by injection of streptozocin (65 mg/kg, i.v.) and insulin was administered via an osmotic minipump to achieve moderate hyperglycemia. Sham rats received vehicle treatments. 2 wk later, the in vitro blood-perfused juxtamedullary nephron technique was used to allow videomicroscopic measurement of afferent arteriolar contractile responses to increasing bath concentrations of either Bay K 8644 or K+. Baseline afferent arteriolar diameter in kidneys from diabetic rats (26.4+/-1.2 microm) exceeded that of Sham rats (19.7+/-1.0 microm). Bay K 8644 evoked concentration-dependent reductions in afferent diameter in both groups of kidneys; however, arterioles from Sham rats responded to 1 nM Bay K 8644 while 100 nM Bay K 8644 was required to contract arterioles from diabetic rats. The EC50 for K+-induced reductions in afferent arteriolar diameter was greater in diabetic kidneys (40+/-4 mM) than in kidneys from Sham rats (28+/-4 mM; P < 0.05). In afferent arterioles isolated by microdissection from Sham rats and loaded with fura 2, increasing bath [K+] from 5 to 40 mM evoked a 98+/-12 nM increase in intracellular Ca2+ concentration ([Ca2+]i). [Ca2+]i responses to 40 mM K+ were suppressed in afferent arterioles from diabetic rats (delta = 63+/-5 nM), but were normalized by decreasing bath glucose concentration from 20 to 5 mM. These observations indicate that the early stage of insulin-dependent diabetes mellitus is associated with a functional defect in afferent arteriolar L-type calcium channels, an effect which may contribute to suppressed afferent arteriolar vasoconstrictor responsiveness and promote glomerular hyperfiltration.     

Altered calcium regulation and function of human neutrophils during multiple trauma

Altered intracellular Ca2+ concentration is a pivotal regulatory mechanism of leukocyte function. Since polymorphonuclear neutrophils (PMN) are involved in traumatic organ dysfunction, we prospectively investigated Ca2+ regulation and function of circulating PMN multiple trauma patients (Group A: ISS < 27; Group B: ISS > or = 27). Circulating PMN were isolated during 12 days, followed by determination of formyl-methionyl-leucyl-phenylalanine (fMLP)-induced PMN-superoxide production (PMN-SOP) by SOD-inhibitable ferricytochrome C reduction, and PMN cytosolic Ca2+ concentration ([Ca2+]i) by fluorescent fura2/AM (340/380 ratio). PMN-SOP was significantly higher in Group B (mean ISS: 39.9 +/- 2; n = 21) at day of admission than in controls and Group A (mean ISS: 18.2 +/- 1; n = 22) (P< 0.05). In Group B, the significant rise of basal [Ca2+]i between Day 2 and Day 4 was associated with significant lower PMN-SOP during that period (P < 0.05). The fMLP-induced [Ca2+]i response was supranormal in both groups. PMN-elastase concentrations were substantially higher in Group B compared with Group A until Day 4. Circulating IL-6, IL-8, and soluble TNF-receptor (55 kD) were significantly increased in Group B compared with Group A at the day of trauma (P < 0.05). Severe trauma is characterized by a biphasic pattern of neutrophil priming characterized by early increase and secondary suppression. The association of depressed neutrophil superoxide production (deactivation) and elevated basal [Ca2+]i suggests Ca2+-mediated disturbance of neutrophil NADPH-oxidase metabolism.     

Interaction of atriopeptin III and vasopressin on calcium kinetics and contraction of aortic smooth muscle cells.

The cellular mechanism of the vasodilatory action of atriopeptin III (APIII) on vasopressin (AVP)-induced Ca2+ mobilization and cell shape change in cultured vascular smooth muscle cells (VSMC) was studied. APIII (10(-8) M) attenuated the increase of intracellular free Ca2+, [Ca2+]i, induced by 10(-8) M AVP (234.0 +/- 14.8 vs. 310.0 +/- 28.4 nM, P less than 0.01). Similar results were obtained in 45Ca2+ efflux experiments. APIII (10(-7) M), however, did not alter AVP-induced inositol trisphosphate (IP3) production, although the levels of inositol-1-phosphate were significantly reduced. The effect of APIII to block or attenuate AVP-induced Ca2+ mobilization was associated with an inhibition of AVP-stimulated cell shape change. The effect of atrial natriuretic factor (ANF) on cell shape, however, occurred at lower ANF concentrations than the effect on the Ca2+ mobilization. APIII stimulated production of cyclic guanosine monophosphate (cGMP) in VSMC. The effect of APIII on AVP-stimulated Ca2+ mobilization was partially mimicked by the stable nucleotide 8-bromo cGMP and was not affected by the soluble guanylate cyclase inhibitor, methylene blue (10(-4) M). These results suggest that APIII exerts its vasodilatory effect, in part, by interference with vasopressor-stimulated Ca2+ mobilization in vascular smooth muscle cells, perhaps by stimulating particulate guanylate cyclase and cGMP. However, an effect of ANF on the contractile mechanism at a site independent of Ca2+ release is also suggested by the present results.     

血必净对脓毒性急性肾损伤大鼠肾小管细胞凋亡和相关蛋白表达的影响

目的探讨早期应用血必净对脓毒性急性肾损伤大鼠肾小管细胞凋亡,及相关蛋白B细胞淋巴瘤2（Bcl-2）和Bcl-2相关X蛋白（Bax）表达的影响。 方法54只雄性大鼠分为假手术组（Sham组）、脓毒症组（CLP组）、血必净组（CLP + XBJ组）三组,每组18只;以术后12、24和48 h作为时间观察点,将各组再分为3个亚组,每组6只。在各时间点检测大鼠的肌酐清除率（CrCl）、肾脏血流灌注量,并留取肾脏组织观察病理学变化,计算肾小管损伤评分,采用原位末端标记（TUNEL）法并通过计算积分光密度值（IOD）测定肾小管凋亡细胞,采用Western-blotting法测定肾脏髓质Bcl-2、Bax的变化。 结果三组大鼠的CrCl和肾脏血流灌注量在术后各时间点差异均有统计学意义（F = 6.405、18.821,P < 0.05）;且CLP + XBJ组在术后48 h时CrCl和肾脏血流灌注量明显高于CLP组[（2.1 ± 0.5）ml/min/100 g vs.（1.1 ± 0.3）ml/min/100 g,（159 ± 38）BPU vs.（79 ± 32）BPU;P均< 0.05]。三组大鼠肾小管损伤评分比较,不同时间点差异存在统计学意义（F = 5.461,P < 0.05）;且CLP + XBJ组与CLP组比较,24、48 h时评分均有显著下降[（1.6 ± 0.5）vs.（2.8 ± 0.8）,（1.8 ± 0.8）vs.（3.6 ± 0.6）;P均< 0.05]。三组大鼠各时间点凋亡细胞IOD值,差异存在统计学意义（F = 7.259,P < 0.05）;CLP + XBJ组各时间点的IOD值均明显小于CLP组[（26.6 ± 6.9）vs.（34.4 ± 5.0）,（38.2 ± 5.3）vs.（48.0 ± 5.8）,（37.6 ± 2.2）vs.（53.8 ± 6.7）;P均< 0.05]。同时,CLP + XBJ组能升高大鼠肾脏髓质Bcl-2的表达,抑制Bax的表达,维持Bcl-2/Bax的平衡,24、48 h时其Bcl-2[（1.30 ± 0.09）vs.（0.76 ± 0.09）,（2.12 ± 0.38）vs.（0.51 ± 0.07）;P均< 0.05]和Bax[（1.19 ± 0.37）vs.（1.95 ± 0.90）,（1.48 ± 0.15）vs.（2.69 ± 0.39）;P均< 0.05]的表达水平与脓毒症组相比差异均有统计学意义。 结论早期应用血必净可以减少肾小管细胞在脓毒症进程中的凋亡,改善脓毒性急性肾损伤大鼠肾脏的病理学改变,维持Bcl-2/Bax的平衡,减轻脓毒症导致的肾功能损害。     

Effects of FK506 on [Ca2+]i differ in mouse and rabbit ventricular myocytes

FK506 binding proteins (FKBPs 12 and 12.6) interact with ryanodine receptor (RyR) and modulate its functions. FK506 binds to and reverses effects of FKBP on RyR, thus increasing RyR sensitivity to Ca2+, decreasing RyR cooperativity, and increasing RyR open probability. FK506 would thus be expected to have an effect on excitation-contraction coupling, but which of these FK506 effects predominates and how the [Ca2+]i transient would be altered are difficult to predict. FK506 has been reported to increase the [Ca2+]i transient in rat myocytes, but effects in other species have not been described. We compared the effects of FK506 on [Ca2+]i transients, L-type Ca2+ channel and Na/Ca exchange currents, membrane potential, and sarcoplasmic reticulum (SR) Ca2+ content in adult mouse and rabbit ventricular myocytes (VM). FK506 (10 microM) increased the [Ca2+]i transient in mouse VM (656 +/- 116 to 945 +/- 144 nM, p < 0.001) but decreased the amplitude of [Ca2+]i transients in rabbit VM (627 +/- 61 to 401 +/- 37 nM, p < 0.001). Similar effects were observed with rapamycin. The effects of FK506 and rapamycin on [Ca2+]i transients in VM of both species were reversible upon washout. FK506 did not alter SR Ca2+ content in mouse VM (0.79 +/- 0.1 versus 0.78 +/- 0.1 pC/pF) but reduced the SR Ca2+ content in rabbit VM (0.43 +/- 0.05 versus 0.30 +/- 0.04 pC/pF, P < 0.05) [pC = the integral (pA. s) of the caffeine-induced inward I(Na/Ca) normalized by cell capacitance (pF)]. FK506 had no effects on membrane potential, I(Ca,L) and outward I(Na/Ca) in either mouse or rabbit VM. These results indicate that alteration of the functions of RyR by FK506-mediated dissociation of FKBP from RyR has different species-dependent effects on SR Ca2+ load and thus [Ca2+]i transients. This difference may result from the fact that [Na+]i is low in rabbit myocytes, allowing extrusion by Na+/Ca2+ exchange of Ca2+ released by FK506-induced dissociation of FKBP12.6 from SR RyR.     

Effect of cortisol-synthesis inhibition on endotoxin-induced porcine acute lung injury, shock, and nitric oxide production

In the process of developing a model of Escherichia coli endotoxin-induced acute lung injury and shock in specific pathogen-free pigs, the effects of pretreatment with metyrapone (a cortisol-synthesis inhibitor) were examined. Metyrapone was administered 1.5 h before start of endotoxin infusion at t = 0 h (MET-ETOX group, n = 6). At the end of the experiments (t = 4 h) a bronchoalveolar lavage (BAL) was performed. Control animals received only endotoxin (CON-ETOX group, n = 6) or metyrapone (MET-CON group, n = 4). The following results are presented as means +/- SEM. It was found that metyrapone successfully blocked endogenous cortisol synthesis (plasma cortisol levels were 41.0 +/- 5.9 nM in MET-ETOX vs. 339.0 +/- 37.7 nM in CON-ETOX at t = 4 h, P <0.01). At t = 4 h the MET-ETOX animals had substantially increased systemic hypotension compared to the CON-ETOX group (mean arterial pressure 26.7 +/- 4.3 vs. 77.7 +/- 12.2 mmHg, P <0.01), decreased dynamic lung compliance (10.9 +/- 0.7 vs. 13.7 +/- 0.6 ml/cmH2O, P <0.01), increased percentage of BAL neutrophils (28.4 +/- 6.5 vs. 6.6 +/-1.8, P <0.01), pulmonary edema (BAL total protein 0.82 +/- 0.21 vs. 0.42 +/- 0.09 mg/mL, P <0.05), elevated levels of interleukin-8 (1924 +/- 275 vs. 324 +/- 131 pg/mL, P <0.01) and acidosis (pH 7.11 +/- 0.03 vs. 7.23 +/- 0.06, P <0.05). The MET-ETOX group also showed an increased pulmonary hypertension between 2 and 3 h after start of endotoxin infusion and a trend toward significantly increased levels of plasma interleukin-8 (P = 0.052). Arterial pCO2, pO2/FiO2, plasma endothelin-1, plasma TNFalpha, and blood leukocytes were not markedly influenced by the plasma cortisol levels. Nitric oxide production did not seem to be altered by endotoxin infusion in this model, in contrast to other animal studies; this discrepancy could be thought to be due to endotoxin-dosage differences or species differences. It is concluded that if endogenous cortisol production is blocked by metyrapone, the reactions occurring as a result of the endotoxin-induced acute lung injury and shock are greatly enhanced and that therefore pretreatment with metyrapone might be an important addition to this model with specific pathogen-free pigs.     

Electrophysiological effects of prucalopride, a novel enterokinetic agent, on isolated atrial myocytes from patients treated with beta-adrenoceptor antagonists

Prucalopride is a selective 5-hydroxytryptamine type 4 (5-HT4) receptor agonist developed for the treatment of gastrointestinal disorders. The endogenous agonist 5-HT acting via 5-HT4 receptors increases the L-type Ca2+ current (I(CaL)) with potentially proarrhythmic consequences (Pau et al., 2003). The aims of this study were to investigate the effects of prucalopride on I(CaL), action potentials, refractory period, and arrhythmic activity in human atrial myocytes, and to compare these with the effects of 5-HT, using the whole-cell perforated patch-clamp technique. Prucalopride (10(-9) to 10(-4) M) produced a concentration-dependent increase in I(CaL) amplitude, with a maximum response at 10 microM, from -5.3 +/- 0.6 to -10.9 +/- 1.5 pA/pF (p < 0.05; n = 22 cells, 10 patients), without affecting its voltage-dependence. Subsequent application of 10 microM 5-HT further increased I(CaL) to -17.7 +/- 2.8 pA/pF (p < 0.05; n = 16 cells, 9 patients). The increase in I(CaL) by prucalopride, 98 +/- 15%, was significantly smaller than that by 5-HT, 233 +/- 26% (p < 0.05). Prucalopride (10 microM) significantly increased the action potential duration at 50% repolarization (APD50) from 12 +/- 2 to 17 +/- 3 ms (p < 0.05; n = 22 cells, 9 patients). Following washout of prucalopride, 5-HT (10 microM) increased APD50, to a greater extent, from 14 +/- 3 to 32 +/- 7 ms (p < 0.05; n = 11 cells; 8 patients). The APD75, APD90, and effective refractory period were unaffected by prucalopride or 5-HT. Furthermore, 5-HT induced abnormal depolarizations in 27% of the cells studied, whereas prucalopride induced none (p < 0.05). In conclusion, in human atrial cells, prucalopride, at concentrations markedly above those used therapeutically, acted as partial agonist on I(CaL) and APD50, with no effect on late repolarization or refractory period, and was devoid of arrhythmic activity.     

Endothelin receptor-coupling mechanisms in vascular smooth muscle: a role for protein kinase C

Endothelin (ET), a peptide that is released from cultured endothelial cells, is a potent vasoconstrictor that induces characteristically long-lasting contractions. We used the A10 vascular smooth muscle cell (VSMC) line to probe mechanisms underlying ET-induced contractions. Intracellular Ca2+ ([Ca2+]i) and pH were monitored in A10 monolayers using the fluorescent dyes Fura-2 and 2,7-bis-carboxyethyl-5,6-carboxyfluorescein, respectively. Synthetic porcine ET induced rapid and transient increases in [Ca2+]i (EC50 value, 0.75 nM; maximum, approximately 6-fold above basal). External Ca2+ removal did not block the ability of ET (0.5 or 50 nM) to increase initial [Ca2+]i, although [Ca2+]i returned to prestimulus levels faster as compared with that seen in the presence of external Ca2+. Total cell 45Ca2+ content decreased within 30 sec and remained below prestimulus values for at least 20 min (34 +/- 2% decrease after 5 min, n = 3) in ET-stimulated VSMC. ET stimulated a transient rise in inositol trisphosphate formation in [3H]myo-inositol labeled VSMC, peaking in 30 sec (62 +/- 20% increase, n = 3). In contrast, ET-stimulated diacylglycerol formation in [3H]arachidonic acid-labeled VSMC was sustained and biphasic, exhibiting two peaks at 15 sec (41 +/- 16% increase) and at 5 min (75 +/- 7% increase, n = 3). ET (50 nM) also induced an intracellular alkalinization of 0.17 +/- 0.02 (n = 10) pH units above basal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Abnormalities in intracellular calcium regulation and contractile function in myocardium from dogs with pacing-induced heart failure.

24 d of rapid ventricular pacing induced dilated cardiomyopathy with both systolic and diastolic dysfunction in conscious, chronically instrumented dogs. We studied mechanical properties and intracellular calcium (Ca2+i) transients of trabeculae carneae isolated from 15 control dogs (n = 32) and 11 dogs with pacing-induced cardiac failure (n = 26). Muscles were stretched to maximum length at 30 degrees C and stimulated at 0.33 Hz; a subset (n = 17 control, n = 17 myopathic) was loaded with the [Ca2+]i indicator aequorin. Peak tension was depressed in the myopathic muscles, even in the presence of maximally effective (i.e., 16 mM) [Ca2+] in the perfusate. However, peak [Ca2+]i was similar (0.80 +/- 0.13 vs. 0.71 +/- 0.05 microM; [Ca2+]o = 2.5 mM), suggesting that a decrease in Cai2+ availability was not responsible for the decreased contractility. The time for decline from the peak of the Cai2+ transient was prolonged in the myopathic group, which correlated with prolongation of isometric contraction and relaxation. However, similar end-diastolic [Ca2+]i was achieved in both groups (0.29 +/- 0.05 vs. 0.31 +/- 0.02 microM), indicating that Cai2+ homeostasis can be maintained in myopathic hearts. The inotropic response of the myopathic muscles to milrinone was depressed compared with the controls. However, when cAMP production was stimulated by pretreatment with forskolin, the response of the myopathic muscles to milrinone was improved. Our findings provide direct evidence that abnormal [Ca2+]i handling is an important cause of contractile dysfunction in dogs with pacing-induced heart failure and suggest that deficient production of cAMP may be an important cause of these changes in excitation-contraction coupling.