Outcome of diabetic foot infections treated conservatively: a retrospective cohort study with long-term follow-up.

BACKGROUND: Diabetic foot lesion is associated with increased morbidity and high resource use. Although early amputation has been advocated in case of osteomyelitis, conservative treatment is a more attractive alternative. OBJECTIVE: To identify criteria predictive of failure of conservative treatment of diabetic foot ulcer at time of admission to the hospital. METHODS: We conducted a 5-year retrospective cohort study with prospective long-term follow-up of all diabetic patients admitted for a foot lesion at a large (1600-bed) teaching institution. Predetermined criteria were used for the diagnosis and classification of diabetic foot lesions (Wagner classification). Study variables included patient demographics and clinical parameters related to infection and diabetes. The average follow-up after hospital discharge was 2 years. Failure of conservative treatment was the main outcome measure. Independent predictor variables were selected by logistic regression analysis. RESULTS: A total of 120 diabetic patients were admitted for foot lesions; complications of contiguous osteomyelitis, deep tissue involvement, and/or gangrenous lesions occurred in 78 (74%) of the 105 patients for whom charts were available. Fourteen patients (13%) underwent immediate amputation. Conservative treatment was successful for 57 (63%) of the 91 remaining patients. Success was achieved in 21 (81%) of 26 patients presenting with skin ulcer, 35 (70%) of 50 patients with deep tissue infection or suspected osteomyelitis, and 1 (7%) of 15 patients with gangrene (P<.001, chi2 for trend). Independent factors predictive of failure were the presence of fever (odds ratio [OR]=1.1 per degrees Celcius; 95% confidence interval [CI], 1.0-1.2) and increased serum creatinine level (OR=1.002 per micromoles per liter; 95% CI, 1.0020-1.0021) on admission, prior hospitalization for diabetic foot lesion (OR=1.4; 95% CI, 1.2-1.6), and gangrenous lesion (OR=1.8; 95% CI, 1.5-2.2). Other patient characteristics, demographics, duration of diabetes mellitus, neutrophil count, or the anatomical site of the lesion failed to predict outcome. CONCLUSIONS: Conservative treatment, including prolonged, culture-guided parenteral and oral antibiotics, is successful without amputation in a large proportion of diabetic patients admitted for a foot skin ulcer or suspected osteomyelitis. Future studies comparing early amputation with novel therapeutic strategies for severe diabetic foot infection should take into account currently identified factors that predicted failure of conservative treatment on admission to the hospital.

Invasive aspergillosis. Clinical features of 35 proven cases at a single institution.

Thirty-five patients with clinical features and histologically or microbiologically proven infection met predetermined stringent criteria for invasive aspergillosis over a 5-year period at our institution. Underlying conditions included hematologic malignancy, solid tumor, bone marrow and solid organ transplantation, and immunosuppressive therapy. The majority of patients (94%) presented with respiratory symptoms and abnormal pulmonary chest radiography; only 40% had neutropenia at time of infection. Invasive aspergillosis was suspected in only 21 cases (60%). Concomitant infections were present in 83% of patients. Half of patients had pathogenic or potentially pathogenic microorganisms other than Aspergillus spp. isolated from pulmonary specimens at time of aspergillosis. Aspergillus spp. were recovered from sputum in 75% of patients and from bronchoalveolar lavage in only 52%. Invasive aspergillosis is an unexpectedly unrecognized disease with poor outcome; overall mortality was 94% in our series. The lack of sensitivity of diagnostic procedures, together with the high frequency of concomitant infections, delays the time of diagnosis. Early diagnostic tests are needed, and presumptive antifungal therapy among high-risk patients is mandatory.

Role of plasma and extracellular matrix proteins in the physiopathology of foreign body infections.

Foreign body implants are highly susceptible to microorganism infection. The infectious agents may be of low pathogenicity (such as S. epidermidis) or involve more virulent strains (such as S. aureus). The common denominator for the three main elements that play a role in the physiopathology of such infections (bacteria, neutrophils, and different biomaterials) are host proteins deposited over the surface of the devices immediately after their implantation. These proteins modulate that host cells response but can also promote Staphylococcus adhesion to the biomaterial. Neutrophils and other cells such as fibroblasts adhere to several extracellular matrix proteins such as fibronectin, fibrinogen, collagen, vitronectin, via specific cell surface receptor. The evolution of the technology and the increasing numbers of long-term artificial implants require a better understanding of fundamental mechanisms of foreign body infections to reduce their incidence and optimize their treatment.

Cytosolic free Ca2+ signals in single adherent human neutrophils: generation and functional role.

To study the role of cytosolic free calcium, [Ca2+]i, in cell activation, in particular during adhesion and movement on a surface in response to chemotactic peptide stimulation and during phagocytosis, we monitored [Ca2+]i in single human neutrophils. The neutrophils were loaded with fura-2 and allowed to adhere to albumin-coated glass coverslips. [Ca2+]i was monitored with a dual excitation microfluorimeter. Half of the cells showed spontaneous [Ca2+]i transients that lasted up to 15 min with an amplitude averaging 77 +/- 10 nM above basal levels (mean basal value of 110 +/- 20 nM) and a mean duration of 28 +/- 5 s. These repetitive [Ca2+]i elevations depended on the continuous presence of extracellular Ca2+ and could be dissociated from those triggered by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP). Cell morphology was monitored in parallel by recording fluorescent images with a high sensitivity charge coupled device (CCD) camera. The majority of the cells studied showed visible changes in shape which started either before or at the same time as the onset of the [Ca2+]i transients. Removal of extracellular Ca2+ abolished [Ca2+]i transients without impairing cell movement and spreading. Blockade of adherence and cell movement with cytochalasin B markedly inhibited [Ca2+]i transients. Monoclonal antibodies directed against the leucocyte integrin CR3 (CD11b/CD18 alpha m beta 2) blocked adherence, spreading and most of the [Ca2+]i activity. Total [Ca2+]i activity was assessed during phagocytosis of C3bi-opsonized yeast particles and correlated with fusion of secondary granules with the phagosomal membrane (P-L fusion).(ABSTRACT TRUNCATED AT 250 WORDS)

InsP3 and Ins(1,3,4,5)P4 act in synergy to stimulate influx of extracellular Ca2+ in Xenopus oocytes.

To investigate the role of D-myo-inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] in the regulation of Ca2+ influx, we injected inositol phosphates into Xenopus oocytes and measured Ca(2+)-gated Cl- current to assay intracellular free Ca2+ concentration ([Ca2+]i). To assess Ca2+ influx, we removed extracellular Ca2+ or added the inorganic Ca2+ channel blocker Mn2+ to the extracellular bath and measured the resulting change in Cl- current. Ins(1,3,4,5)P4 did not cause Ca2+ influx when injected alone or when preceded by an injection of Ca2+. In contrast, Ins(1,3,4,5)P4 stimulated Ca2+ influx when injected after the poorly metabolized inositol trisphosphate (InsP3) analogues D-myo-inositol 1,4,5-trisphosphorothioate [Ins(1,4,5)P3S3] or D-myo-inositol 2,4,5-trisphosphate [Ins(2,4,5)P3]. These results indicate that Ins(1,3,4,5)P4 is not sufficient to stimulate Ca2+ influx but acts in synergy with InsP3s to cause Ca2+ influx. We also studied the effect of Ca2+ influx on the immediate metabolism of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in single oocytes. Ca2+ influx shunted the metabolism of Ins(1,4,5)P3 toward the formation of Ins(1,3,4,5)P4 and away from D-myo-inositol 1,4-bisphosphate [Ins(1,4)P2]. These results suggest that there is a positive feedback regulatory mechanism in which Ca2+ influx stimulates Ins(1,3,4,5)P4 production and Ins(1,3,4,5)P4 stimulates further Ca2+ influx.

The chymotrypsin inhibitor carbobenzyloxy-leucine-tyrosine-chloromethylketone interferes with the neutrophil respiratory burst mediated by a signaling pathway independent of PtdInsP2 breakdown and cytosolic free calcium.

The effects of carbobenzyloxy-leucine-tyrosine-chloromethylketone (zLYCK), an inhibitor of chymotrypsin, were investigated on the activation pathways of the human neutrophil respiratory burst. At 10 microM zLYCK, a parallel inhibition was observed of superoxide production stimulated with the chemo-attractant FMLP and of chymotrypsin-like activity of human neutrophils. By contrast, superoxide production induced by PMA was minimally affected by zLYCK. The known transduction pathways triggered by FMLP were analyzed. zLYCK did not affect either the FMLP-induced cytosolic free calcium transient, inositol 1,4,5 trisphosphate formation, nor the PMA-induced phosphorylation of the 47-kDa substrate of protein kinase C. zLYCK did not affect the activity of protein kinase C extracted from neutrophils. In Ca(2+)-depleted cells, in which phosphatidylinositol 4,5-biphosphate breakdown does not occur, zLYCK inhibited the FMLP-induced respiratory burst in cells primed by low doses of PMA. The activity of the NADPH oxidase tested with active membranes from stimulated neutrophils or in a cell-free system was not inhibited by zLYCK. We conclude that: 1) zLYCK inhibits superoxide production through the inhibition of a chymotrypsin-like protease of the neutrophil, 2) zLYCK inhibits FMLP-induced activation of NADPH oxidase through a pathway independent of PtdInsP2 breakdown and cytosolic free calcium, and 3) zLYCK may prove a useful probe for the characterization of its target protease in neutrophil activation.

Perioperative antibiotic prophylaxis of wound and foreign body infections: microbial factors affecting efficacy.

Numerous microbial factors are responsible for perioperative infections and influence the efficacy of antibiotic prophylaxis. These factors include the staphylococcal carrier state, bacterial adherence to a number of host proteins, the production of glycocalyx by sessile bacteria, and shifts in antibiotic resistance. A full understanding of the mechanisms involved will lead to further reductions in the number of postoperative infections. Unfortunately, the microbial factors affecting prophylaxis cannot be evaluated separately under clinical conditions; they are easier to study under circumstances whose bacteriologic features are well defined and in which the presence of foreign materials (e.g., sutures) greatly potentiates pathogenic mechanisms. Such circumstances exist, for example, in infections developing after "clean" surgery and in experimental models. Since even clean wounds are found to be contaminated when sampled carefully, the control of infection is more a quantitative than a qualitative problem. The critical period for the development of infection is short: an antibiotic course not exceeding 24 hours seems effective in preventing infection.

The chymotrypsin inhibitor carbobenzyloxy-leucine-tyrosine-chloromethylketone interferes with phospholipase D activation induced by formyl-methionyl-leucyl-phenylalanine in human neutrophils.

The effects of carbobenzyloxy-leucine-tyrosine-chloromethylketone (zLYCK), an inhibitor of chymotrypsin-like proteases, on signal transduction in human neutrophils triggered by the chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP) were investigated. zLYCK (10 microM) inhibited the fMLP-induced respiratory burst in neutrophils treated with cytochalasin B. In the presence of zLYCK (10 microM), the activation of phospholipase D in response to fMLP addition was inhibited. zLYCK did not inhibit the binding of [3H] fMLP to its receptor or the enzymic activity of phospholipase D because the response to ionomycin was unaffected. The effect of zLYCK on phospholipase D correlated well with its effects on the accumulation of diglycerides, which was also inhibited in the presence of zLYCK. In electropermeabilized neutrophils, too, zLYCK caused an inhibition of the fMLP-induced respiratory burst and the fMLP-induced activation of phospholipase D. Interestingly, this inhibition could be bypassed by guanosine 5'-O-(thiotriphosphate). We conclude that the inhibition of the respiratory burst in human neutrophils by zLYCK is caused by the selective inhibition of signal transduction leading to activation of phospholipase D and that zLYCK might be a useful probe to study the role of phospholipase D in neutrophil activation.

Diagnosis of ventilator-associated pneumonia by bacteriologic analysis of bronchoscopic and nonbronchoscopic "blind" bronchoalveolar lavage fluid.

Substantial efforts have been devoted to improving the means for early and accurate diagnosis of ventilator-associated (VA) pneumonia in intensive care unit (ICU) patients because of its high incidence and mortality. A good diagnostic yield has been reported from quantitative cultures of bronchoalveolar lavage (BAL) fluid or a protected specimen brush, both obtained by fiberoptic bronchoscopy. As bronchoscopy requires specific skills and is costly, we evaluated a simpler method to obtain BAL fluid, that is, by a catheter introduced blindly into the bronchial tree. Quantitative cultures from bronchoscopically sampled BAL (B-BAL) and blindly nonbronchoscopically collected BAL (NB-BAL) were assessed for sensitivity, specificity, and predictive value for the diagnosis of VA pneumonia. A total of 40 pairs of samples were examined in 28 patients requiring prolonged mechanical ventilation and presenting a high risk of developing pneumonia. For comparison with bacteriologic data we defined a clinical score for pneumonia ranging from zero to 12 using the following variables: body temperature, leukocyte count, volume and character of tracheal secretions, arterial oxygenation, chest X-ray, Gram stain, and culture of tracheal aspirate. To quantify the bacteria in BAL the bacterial index (BI) was used, defined as the sum of the logarithm of the number of bacteria cultured per milliliter of BAL fluid. A good correlation between clinical score and quantitative bacteriology was observed (r = 0.84 for B-BAL and 0.76 for NB-BAL; p less than 0.0001). Similar to studies in baboons, patients with pulmonary infection could be distinguished by a BI greater than or equal to 5 with a sensitivity of 93% and a specificity of 100% (B-BAL).(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple elevations of cytosolic-free Ca2+ in human neutrophils: initiation by adherence receptors of the integrin family.

Multiple spontaneous transient elevations of cytosolic-free calcium ([Ca2+]i) are observed in single human neutrophils during adherence. The interrelation between adherence and spontaneous [Ca2+]i transients was analyzed by simultaneous monitoring of [Ca2+]i and cell morphology. Fluorescent images of fura 2-loaded neutrophils attached to albumin-coated glass were recorded with a high sensitivity CCD camera while [Ca2+]i was assessed with a dual excitation microfluorimetry. The majority of the initially round cells studied showed changes in shape which started either before or at the same time as the onset of the [Ca2+]i transients. These data suggested that a rise in [Ca2+]i is not a prerequisite for shape change. This conclusion was confirmed by observation of movement and spreading in cells whose [Ca2+]i transients were abolished by chelation of extracellular Ca2+. Instead, our data suggest that spreading or adhesion itself initiates the [Ca2+]i activity. In keeping with this hypothesis, cytochalasin B, which prevents both cell movement and adhesion, completely inhibited generation of [Ca2+]i transients. To determine if the movement alone or adhesion alone is responsible for [Ca2+]i activity, we treated cells with antibodies against the beta chain (CD18, beta 2) or the alpha subunit (CD11b, alpha m) of the dominant leukocyte integrin (CR3). Antibody-treated cells showed normal extension of pseudopods but impaired ability to adhere. Inhibition of adhesion in this way inhibited [Ca2+]i activity. Taken together these results suggest that following sequence of events after contact of neutrophils with surfaces: (a) cell movement and shape change lead to enhanced contact of integrins with the surface; and (b) integrins-mediated adhesion generates multiple [Ca2+]i transients. The [Ca2+]i transients may then control exocytic events associated with movement and may provide a link between adherence and activation or priming of neutrophils to other stimuli.

Thrombospondin binds to Staphylococcus aureus and promotes staphylococcal adherence to surfaces.

Bacterial adherence to surfaces is the determining first step in staphylococcal infections. Activated platelets mediate adherence of staphylococci to tissues during inflammation or infection; however, the molecular mechanisms of this interaction are not clearly understood. Thrombospondin, a large multifunctional glycoprotein, is the principal platelet-stored glycoprotein. It is secreted upon platelet activation and either bound to receptors on the platelet surface or released and incorporated into blood clots and extracellular matrices. To characterize thrombospondin binding to staphylococci, we incubated [125I]thrombospondin with Staphylococcus aureus Cowan 1 in the presence of albumin and separated bound and free thrombospondin by centrifugation. We found that binding was (i) specific, since it was up to 76% inhibitable and up to 60% reversible in the presence of a 100-fold excess of unlabeled thrombospondin, (ii) saturable, with an apparent dissociation constant (Kd) of 5.6 x 10(-9) M and a maximal number of 2,600 binding sites per microorganism, and (iii) Ca2+ dependent, since omission of this ion from the medium decreased significantly the binding capacity. The binding reaction was insensitive to previous trypsin treatment of bacteria, but it was strongly inhibited in the presence of heparin. Protein A-negative and -positive strains had similar binding characteristics. To determine the promotion of staphylococcal adherence to surfaces by solid-phase thrombospondin, we incubated 3H-labeled S. aureus Cowan 1 and 26 pathogenic staphylococcal isolates with thrombospondin-coated polymethylmethacrylate disks and found that adherence was significantly promoted as a function of adsorbed thrombospondin. These results indicate a role for thrombospondin as an important mediator of staphylococcal adherence to activated platelets, to blood clots, or to extracellular matrices in pyogenic infections.

Receptors and intracellular signaling in human neutrophils.

Adherence, chemotaxis, phagocytosis, and responses to cytokines are mediated by distinct classes of cell surface receptors in human neutrophils. Intracellular signaling by these different receptors is a subject of active investigation. Observation of single neutrophils adherent to surfaces reveals the presence of spontaneous oscillations of cytosolic-free calcium, [Ca2+]i, generated by mechanisms that are presently unknown. Chemoattractant receptor activation via a specific G-regulatory protein activates a plasma membrane phospholipase C and generates diacylglycerol and inositol(1,4,5)triphosphate. DG activates C kinase(s). Ins(1,4,5)P3 releases Ca2+ from a specific intracellular organelle, the calciosome. Calciosomes resemble sarcoplasmic reticulum: they contain a Ca2(+)-ATPase and a high capacity/low affinity calcium-binding, calsequestrin-like protein. Chemoattractant receptor stimulation of calcium influx across the plasma membrane in phagocytes correlates strongly with the conversion of Ins(1,3,4,5)P3 to Ins(1,3,4,5)P4 by a Ca2(+)-calmodulin-sensitive kinase. The transduction system of phagocytosis receptors also generates DG and Ins(1,4,5)P3 and elicits [Ca2+]i elevations. The Ca2+ signal is an important regulator of secretion (granule exocytosis, superoxide production), whereas C kinase(s)/and other unknown mediators appear to be more important for the control of movement. Several mechanisms that could account for the specificity of cell signaling by different receptors are discussed.

Inhibition by immunoglobulins of Staphylococcus aureus adherence to fibronectin-coated foreign surfaces.

Recent data suggest that fibronectin may favor Staphylococcus aureus infection by promoting attachment to either injured tissues or implanted foreign bodies. Using a previously described in vitro assay, we show that promotion of S. aureus adherence by surface-bound fibronectin, adsorbed on polymethylmethacrylate (PMMA) coverslips, is antagonized by antistaphylococcal antibodies present in immunoglobulin G (IgG) purified from human plasma. Among the different organisms tested, the protein A-deficient strain Wood 46 of S. aureus was the most strongly inhibited by purified IgG or whole serum dose-dependently. Bacterial adherence was not influenced by preincubating fibronectin-coated PMMA with either purified IgG or whole serum. However, inhibition of bacterial adherence was directly related to the extent of IgG binding to S. aureus Wood 46. When F(ab')2 fragments of purified IgG were tested in the adherence assay, they could also reduce the interaction between S. aureus Wood 46 and fibronectin-coated PMMA. Two other staphylococcal strains were also tested in the adherence inhibition assay: Whereas the protein A-rich strain Cowan I of S. aureus was moderately inhibited by purified IgG or whole serum, S. epidermidis KH 11 was not at all inhibited by IgG which bound poorly to the bacterial cells. This study has demonstrated that bacterial coating by humoral factors, and specifically IgG, may influence significantly subsequent adherence of S. aureus to surface-bound fibronectin.

Fibronectin, fibrinogen, and laminin act as mediators of adherence of clinical staphylococcal isolates to foreign material.

Bacterial adherence to polymer surfaces is a required early step in intravenous (iv) device infection. We collected eight strains of Staphylococcus aureus and 19 of coagulase-negative staphylococci from patients with proven iv device bacteremia and studied the role of plasma or connective-tissue proteins in promoting bacterial adherence to polymethylmethacrylate (PMMA) coverslips. Although only a negligible percentage of organisms adhered to albumin-coated PMMA, surface-bound fibronectin significantly promoted adherence of all isolates. Fibrinogen markedly promoted adherence of all S. aureus strains but of only four coagulase-negative strains. Thus, coagulase-negative staphylococci revealed a marked heterogeneity in adherence to fibrinogen-coated surfaces, a result suggesting the existence of heretofore unknown receptors for fibrinogen. Laminin promoted adherence of staphylococci to a much lower extent. Although strain specific, adherence of clinical staphylococcal isolates to foreign surfaces is significantly increased by fibronectin, fibrinogen, and laminin, an observation suggesting the possible contribution of these proteins to the pathogenesis of iv device infection.

Spontaneous and chemoattractant-induced oscillations of cytosolic free calcium in single adherent human neutrophils.

Studies with fluorescent Ca2+ indicators in large populations of neutrophils in suspension reveal a stable base line followed by a rapid agonist-induced elevation of cytosolic free calcium, [Ca2+]i, concomitant with other parameters of cellular activation. To study the role of adhesion in cell activation, we monitored [Ca2+]i in single neutrophils adhered to albumin-coated or fibronectin-coated glass coverslips before and after stimulation with the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). Human neutrophils loaded with 2 microM fura 2/AM were allowed to adhere to coverslips for 15-20 min at 37 degrees C. [Ca2+]i was monitored with a dual excitation microfluorimeter with a time resolution of 200 ms. Statistical analysis was performed using an algorithm allowing to detect significant [Ca2+]i peaks. 54% of the cells showed spontaneous [Ca2+]i oscillations. The amplitude of these [Ca2+]i peaks averaged 77 +/- 10 nM above basal levels (mean value of 110 +/- 20 nM), and their mean duration was 28 +/- 5 s; periods of [Ca2+]i bursts could last up to 15 min. In "silent" cells exhibiting a stable [Ca2+]i base line without spontaneous oscillations, low concentrations of fMLP (10(-10)-10(-9) M) could induce sustained [Ca2+]i oscillations. By contrast, higher agonist concentrations (10(-6) M) induced a single [Ca2+]i transient followed by a stable base line. 47% of the cells showing spontaneous [Ca2+]i oscillations did not respond to fMLP. Spontaneous [Ca2+]i oscillations depended on the continuous presence of extracellular Ca2+. Therefore: (i) spontaneous oscillations of [Ca2+]i occur in neutrophils adherent to various substrata; (ii) these oscillations do not preclude and can be dissociated from the response to fMLP; (iii) neutrophil functions might be controlled by [Ca2+]i oscillations rather than by sustained alterations of [Ca2+]i.

Effect of serotonin on cytosolic free calcium in adrenal glomerulosa and vascular smooth muscle cells.

In view of its vasoconstricting action and of its stimulating effect on aldosterone biosynthesis, serotonin (5-hydroxytryptamine, 5-HT) could play a role in the genesis and/or maintenance of hypertension. The effects are mediated by different specific receptors whose transmembrane signaling system is not elucidated. We have used the fluorescent probe quin 2 to study the effect of 5-HT on cytosolic free calcium in enzymatically dispersed bovine adrenal glomerulosa cells and in cultured rat aortic smooth muscle cells. We also examined the effect of 5-HT on prostacyclin production by rat aortic smooth muscle cells. Serotonin did not modify the level of cytosolic free calcium in adrenal glomerulosa cells. In contrast, serotonin induced rapid, concentration-dependent (10(-8) -10(-5) M) rises of cytosolic free calcium in monolayers of cultured rat aortic smooth muscle cells, from a basal level of 153 +/- 27 nM to peak levels of about 400 nM. Ketanserin (10(-6) M), a specific 5-HT2-receptor antagonist completely blocked the free calcium rise induced by 5-HT. 5-HT induced a concentration-dependent increase in 6-keto-PGF1 alpha production in smooth muscle cells, which was suppressed by ketanserin, indomethacin or removal of calcium from the incubation medium. In contrast nifedipine (10(-6) M) did not modify the response to 5-HT while it abolished the response to vasopressin and did not modify the response to angiotensin II. We conclude that the 5-HT receptors of adrenal glomerulosa cells and vascular smooth muscle cells are linked to two distinct signalling systems which mediate the different biological responses.

Quantitative analysis of the cytosolic-free-Ca2+-dependency of aldosterone production in bovine adrenal glomerulosa cells. Different requirements for angiotensin II and K+.

Angiotensin II (AII) and K+ raise the cytosolic free Ca2+ concentration [( Ca2+]i) and stimulate aldosterone production in isolated bovine adrenal glomerulosa cells. The mechanisms leading to an elevation of [Ca2+]i were analysed with the fluorescent Ca2+ probe quin 2. (1) Whereas [Ca2+]i rose transiently and returned to basal values within 5 min in response to AII, the effect of K+ was sustained for at least 15 min. (2) AII released Ca2+ from intracellular stores, whereas the [Ca2+]i response to K+ depended solely on extracellular [Ca2+]. (3) When added after K+ stimulation, AII provoked a dramatic decrease in [Ca2+]i to below the resting value. The role of [Ca2+]i in stimulating steroidogenesis was determined by manipulating the concentration of this cation. (4) In a cell superfusion system, the aldosterone response to AII is biphasic. Suppressing the transient [Ca2+]i elevation triggered by AII resulted in the disappearance of the initial secretory peak, but the final production rate was similar to that of control cells. (5) Normal basal [Ca2+]i levels were, however, necessary to maintain continuous AII-induced steroidogenesis. (6) When added after AII, the antagonist analogue [Sar1,Ala8]AII suppressed steroidogenesis without affecting [Ca2+]i levels. (7) In contrast, continuously elevated [Ca2+]i values were required for the initiation and the maintenance of K+-stimulated aldosterone production. These results demonstrate important differences in the mechanisms through which AII and K+ activate the Ca2+ messenger system. Moreover, functional correlations have shown that K+, but not AII, depends solely on a sustained [Ca2+]i response for its steroidogenic effect. However, the AII-induced effect is also a Ca2+-requiring process: the initial [Ca2+]i transient accelerates the onset of steroidogenesis, which is subsequently extremely sensitive to [Ca2+]i decreases below normal basal levels.

Subcellular distribution of Ca2+ pumping sites in human neutrophils.

The distribution of nonmitochondrial Ca2+ pumping sites and the site of action of inositol 1,4,5-trisphosphate (Ins 1,4,5-P3) were studied in subcellular fractions of human neutrophils. In homogenates, two different Ca2+ pools could be observed: a mitochondrial Ca2+ pool and a nonmitochondrial, ATP-dependent, Ins 1,4,5-P3-responsive Ca2+ pool. When the homogenate was separated into microsomes, primary granules, and secondary granules, the nonmitochondrial Ca2+ pumping and the Ins 1,4,5-P3-induced Ca2+ release occurred only in the microsomal fraction. In a gradient developed to separate different microsomal organelles, maximal Ca2+ pumping activity occurred in fractions of low densities. Correlations between Ca2+ uptake and organelle markers were negative for the endoplasmic reticulum (r = -0.49) and positive for plasma membrane (r = 0.47), Golgi (r = 0.62), and endosomes (r = 0.96). Because the Ca2+ pumping organelles in these fractions were insensitive to micromolar vanadate and digitonin treatment, they are unlikely to be plasma membrane vesicles. We conclude first that microsomal fractions of human neutrophils contain organelles that lower the ambient free Ca2+ concentration and respond to Ins 1,4,5-P3. Second, granules are not involved in intracellular Ca2+ regulation in neutrophils. Third, nonendoplasmic reticulum organelles, such as endosomes, Golgi elements, or yet undefined specialized structures, play a major role in intracellular Ca2+ homeostasis in human neutrophils.

Plasma membrane potential modulates chemotactic peptide-stimulated cytosolic free Ca2+ changes in human neutrophils.

The relationship between fMet-Leu-Phe-induced changes in the cytosolic free Ca2+ concentration [( Ca2+]i), plasma membrane potential depolarization, and metabolic responses was studied in human neutrophils. Receptor-activated depolarization occurred both at high and resting [Ca2+]i, but was inhibited at very low [Ca2+]i. Phorbol 12-myristate 13-acetate-induced plasma membrane depolarization, on the contrary, was independent of [Ca2+]i. The threshold fMet-Leu-Phe concentration for plasma membrane depolarization (10(-8) M) was at least 1 log unit higher than that for [Ca2+]i increases (5 X 10(-10) M) and coincident with that for NADPH oxidase activation. Nearly maximal [Ca2+]i increases were elicited by 3 X 10(-9) fMet-Leu-Phe in the absence of any significant plasma membrane potential change. This observation allowed us to investigate the effects of artificially induced plasma membrane depolarization and hyperpolarization at low fMet-Leu-Phe concentrations (10(-9) to 3 X 10(-9) M) which did not perturb plasma membrane potential. Depolarizing (gramicidin D at 10(-7) to 10(-6) M or KCl at 50 mM) and hyperpolarizing (valinomycin at 4 microM) treatments had little influence on unstimulated [Ca2+]i levels, whereas fMet-Leu-Phe-induced transients were significantly altered. Gramicidin D and KCl decreased the fMet-Leu-Phe-induced [Ca2+]i increases in Ca2+-containing or in Ca2+-free media. Valinomycin, on the contrary, increased receptor-stimulated [Ca2+]i increases, and the effect was larger in the presence of extracellular Ca2+. Valinomycin also strongly potentiated secretion. It is suggested that plasma membrane depolarization in human neutrophils is a physiological feedback mechanism inhibiting receptor-dependent [Ca2+]i changes.