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At least 19 recordsLinked to original sources
PubMed2000

Pharmacological and immunohistochemical characterization of calmodulin-stimulated (Ca(2+)+Mg(2+))-ATPase in cultured porcine aortic endothelial cells.

Plasma membrane (Ca(2+)+Mg(2+))-ATPase and Ca(2+) transport activities, best characterized in human erythrocytes, are stimulated by calmodulin and thought to play a crucial role in the termination of cellular Ca(2+) signaling in all cells. In plasma membranes isolated from cultured porcine aortic endothelial cells, the (Ca(2+)+Mg(2+))-ATPase was not readily measured. This is in part because of an overabundance of nonspecific Ca(2+)- and/or Mg(2+)-activated ecto-5'-nucleotide phosphohydrolases. Moreover, addition of exogenous calmodulin (10(-9) to 10(-6) mol/L) produced no measurable stimulation of ATPase activities, suggesting a permanently activated state or, alternatively, a complete lack thereof. To establish and verify the presence of a calmodulin-regulated (Ca(2+)+Mg(2+))-ATPase activity in these endothelial cells, immunohistochemical localization using a monoclonal mouse anti-(Ca(2+)+Mg(2+))-ATPase antibody (clone 5F10) was applied to intact pig aorta endothelium, cultured endothelial monolayers, and isolated endothelial plasma membrane fractions. This approach clearly demonstrated Ca(2+) pump immunoreactivity in each of these preparations. To confirm functional calmodulin stimulation of the (Ca(2+)+Mg(2+))-ATPase, 10(-5) mol/L calmidazolium (R24571) was added to the isolated plasma membrane preparation, which lowered the (Ca(2+)+Mg(2+))-ATPase activity from 143.0 to 78.15 nmol P(i)/mg protein x min(-1). This calmidazolium-reduced activity could then be stimulated 113.1+/-0.8% in a concentration-dependent manner by the addition of exogenous calmodulin (10(-7) to 2 x 10(-6) mol/L) with an EC(50) of 3.45+/-0.04 x 10(-7) mol/L (n=4). This represents a competitive lowering of the apparent calmodulin affinity by approximately 100 compared with other unopposed calmodulin-stimulated processes. Together, these findings support evidence for the presence of a calmodulin-stimulated plasma membrane (Ca(2+)+Mg(2+))-ATPase activity in cultured porcine aortic endothelial cells.

Animals↗
PubMed2000

Ca(2+)-Mg(2+)-ATPase activity and ionized calcium in Type 2 diabetic patients with neuropathy.

Ca(2+)-Mg(2+)-ATPase is an important regulator of intracellular calcium concentration and therefore, of erythrocyte deformability. We have investigated the possible relationship between Ca(2+)-Mg(2+)-ATPase activity (ATPase) and ionized calcium (Ca(2+)), in diabetic patients with peripheral neuropathy. A total of 104 Type 2 diabetic patients (57 neuropathic and 47 non-neuropathic) and 25 non-diabetic subjects were studied. After an overnight fast, blood was taken for Ca(2+)-Mg(2+)-ATPase activity, Ca(2+), Mg(2+), PTH and HbA(1c). The neuropathy study group had significantly lower levels of ATPase, 6.6 (95% CI, 5.6-7.7) nmol/mg/min compared to controls 7. 1 (6.2-8.3) nmol/mg/min, P<0.001 and to diabetic patients without neuropathy 7.0 (6.0-8.1) nmol/mg/min, P<0.001. The study group had also lower levels of Ca(2+) (0.89+/-0.18 mmol/l vs. control 1.08+/-0. 24 mmol/l, P<0.01 and non-neuropathic 0.98+/-0.27 mmol/l, P<0.05) and Mg(2+) (0.73+/-0.13 mmol/l vs. control 0.81+/-0.14 mmol/l, P<0. 05), despite similar PTH levels. In diabetic subjects, no correlation was found between ATPase or Ca(2+) with glucose, HbA(1c), age or duration of diabetes. We conclude that in patients with diabetic neuropathy there are abnormalities of Ca(2+)-Mg(2+)-ATPase activity and Ca(2+). This provides further support for the role of microangiopathy in the pathogenesis of neuropathy.

Blood Glucose↗
PubMed1995

Cholesterol oxidation reduces Ca(2+)+MG (2+)-ATPase activity, interdigitation, and increases fluidity of brain synaptic plasma membranes.

These experiments examined effects of cholesterol oxidation on Ca(2+)+Mg(2+)-ATPase activity, Na(+)+K(+)-ATPase activity, and membrane structure of brain synaptic plasma membranes (SPM). Cholesterol oxidase [E.C.1.1.3.6 from Brevibacterium sp.] was used to oxidize cholesterol. Two cholesterol pools were identified in synaptosomal membranes based on their accessibility to cholesterol oxidase. A rapidly oxidized cholesterol pool was observed with a 1t1/2 of 1.19 +/- 0.09 min and a second pool with a 2t1/2 of 38.30 +/- 4.16 min. Activity of Ca(2+)+Mg(2+)-ATPase was inhibited by low levels of cholesterol oxidation. Ten percent cholesterol oxidation, for example, resulted in approximately 35% percent inhibition of Ca(2+)+Mg(2+)-ATPase activity. After 13% cholesterol oxidation, further inhibition of Ca(2+)+Mg(2+)-ATPase activity was not observed. Activity of Na(+)+K(+)-ATPase was not affected by different levels of cholesterol oxidation (5%-40%). SPM interdigitation was significantly reduced and fluidity was significantly increased by cholesterol oxidation. The relationship observed between SPM interdigitation and Ca(2+)+Mg(2+)-ATPase activity was consistent with studies using model membranes [7]. Brain SPM function and structure were altered by relatively low levels of cholesterol oxidation and is a new approach to understanding cholesterol dynamics and neuronal function. The sensitivity of brain SPM to cholesterol oxidation may be important with respect to the proposed association between oxygen free radicals and certain neurodegenerative diseases.

Animals↗
PubMed2006

Post-treatment with the Ca(2+)-Mg(2+)-endonuclease inhibitor aurintricarboxylic acid prevents peroxynitrite-induced DNA damage and death of murine astrocytes.

Oxidative stress plays critical roles in aging, cell death, and many diseases. Peroxynitrite is one of the major reactive oxygen species which mediates cell injury in a number of illnesses. It is of importance to identify the downstream events in peroxynitrite-initiated cell death cascade for preventing peroxynitrite toxicity. Ca(2+)-Mg(2+)-endonucleases have been suggested as the endonucleases that execute DNA fragmentation in several apoptotic cascades. In this study, we determined if astrocytes and neurons express the genes of Ca(2+)-Mg(2+)-endonucleases. We also tested our hypothesis that post-treatment with the Ca(2+)-Mg(2+)-endonuclease inhibitor aurintricarboxylic acid can decrease peroxynitrite-induced DNA damage and death of astrocytes. We found that both astrocytes and neurons express DNase I-like endonuclease-a major isoform of Ca(2+)-Mg(2+)-endonucleases. Treatment of astrocytes with aurintricarboxylic acid either before or after peroxynitrite exposures can profoundly decrease peroxynitrite-induced DNA damage and cell death. These results suggest that Ca(2+)-Mg(2+)-endonucleases may be a key downstream component in peroxynitrite-initiated cell death cascade in astrocytes and some other cell types, and aurintricarboxylic acid could be used to decrease peroxynitrite-induced DNA damage at delayed phases.

Animals↗
PubMed1993

Cell membrane microviscosity and Ca(2+)-Mg(2+)-ATPase activity do not contribute to hypertension in the spontaneously hypertensive rat model.

1. The relationships between systolic blood pressure and altered erythrocyte Ca(2+)-Mg(2+)-ATPase activity and membrane microviscosity were assessed in membranes prepared from 20-week-old female Wistar-Kyoto normotensive and spontaneously hypertensive rats obtained from two different sources (Charles River and Harlan OLAC) and a second filial (F2) generation derived from a cross between Wistar-Kyoto rats and spontaneously hypertensive rats from one source (Charles River). 2. Spontaneously hypertensive rats from both sources had systolic blood pressures significantly higher than those of Wistar-Kyoto animals (P < 0.05; 151 +/- 4 and 110 +/- 3 mmHg, Charles River; 155 +/- 4 and 122 +/- 4 mmHg, Harlan OLAC). The systolic blood pressures for the F2 rat population ranged between 73 and 168 mmHg. 3. Ca(2+)-Mg(2+)-ATPase activity was measured as ATP-dependent 45Ca2+ uptake into inside-out vesicles and microviscosity assessed by the measurement of polarization anisotropy of membrane incorporated fluorescent probes including 1,6-diphenyl-1,3,5-hexatriene, trimethylamino-1,6-diphenyl-1,3,5-hexatriene and a series of anthroyloxy fatty acids. 4. Contrary to previous studies, no relationship between adult systolic blood pressure and erythrocyte Ca(2+)-Mg(2+)-ATPase activity or general or localized membrane microviscosity was indicated by the comparison of spontaneously hypertensive and Wistar-Kyoto animals or in the analysis of the F2 rat population. 5. These results suggest that Ca(2+)-Mg(2+)-ATPase activity and membrane microviscosity are causally unrelated to hypertension in these animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2001

Bile canalicular contraction and dilatation in primary culture of rat hepatocytes--possible involvement of two different types of plasma membrane Ca(2+)-Mg(2+)-ATPase and Ca(2+)-pump-ATPase.

Increasing evidence has indicated that bile canalicular contraction is mediated by the nonmuscular Ca(2+)-calmodulin-actomyosin system, and the contraction facilitates canalicular bile flow. The aim of the present study was to examine, by electron cytochemistry, how the expression of two types of plasma membrane Ca(2+)-ATPase, i.e., Ca(2+)-Mg(2+)-ATPase and Ca(2+)-pump-ATPase, is related to the dynamic changes of bile canalicular contraction. Hepatocytes isolated from male Wistar rat liver by collagenase perfusion were cultured to form a primary monolayer. The canalicular dynamics in the couplets and triplets were analyzed by time-lapse cinematography. The Ca(2+)-Mg(2+)-ATPase activity was identified by the electron cytochemical method of Ando. Ultrastructural localization of Ca(2+)-pump-ATPase was examined by immunogold electron microscopy. We found that cytochemical reaction products showing the presence of Ca(2+)-Mg(2+)-ATPase activity were localized on the luminal side of the bile canalicular membranes. Immunogold particles, indicating the presence of Ca(2+)-pump-ATPase, were located mainly on the cytoplasmic side of the bile canalicular membranes. The expression of both Ca(2+)-ATPases on the canalicular membranes was enhanced during the contracting stage of bile canaliculi, whereas their expression was diminished in the dilating stage. We conclude that two different types of bile canalicular Ca(2+)-ATPase may be involved in the regulation of canalicular contractility to control the extrusion of intracytoplasmic free calcium ions into the canalicular lumen.

Animals↗
PubMed1994

[Effect of verapamil on Ca2+ and Ca(2+) Mg(2+)-ATPase activity in rat brain synaptosomes].

To elicit the correlation between the adrenergic transmitter release and calmodulin (CaM), the effect of verapamil on the free Ca2+ concentration was measured with fluorescence analysis and Ca(2+) Mg(2+)-ATPase activity in rat synaptosomes were studied. When stimulated with high-K+ or norepinephrine, the concentration of free Ca2+ in rat synaptosome was increased by verapamil 10, 50, and 100 mumol.L-1. But the free Ca2+ concentration in the resting synaptosome was reduced by verapamil. The activity of Ca(2+) Mg(2+)-ATPase in synaptosome was remarkably inhibited by verapamil in a dose-dependent manner. These results support our hypothesis that CaM not only acts directly on the vesicles to enhance the transmitter release, but also acts on the activity of Ca(2+) Mg(2+)-ATPase to reduce the free Ca2+ in the cytosol, and indirectly inhibited the transmitter release.

Animals↗
PubMed1997

[Effect of xianzhen tablet on Na(+)-K(+)-ATPase, Ca(2+)-Mg(2+)-ATPase, of red blood cell membrane in patients with non-insulin dependent diabetes mellitus].

OBJECTIVE: To assess the effect of Xianzhen tablet in treating non-insulin dependent diabetes mellitus (NIDDM). METHODS: Seventy-two cases of NIDDM patients with deficiency of both Qi and Yin, deficiency of the Kidney and blood stasis were selected, and the effects of treatment on Na(+)-K(+)-ATPase, Ca(2+)-Mg(2+)-ATPase, whole blood viscosity, blood sugar and clinical symptoms were observed. RESULTS: After treatment, the treated group showed increased activities that were statistically significant of Na(+)-K(+)-ATPase and Ca(2+)-Mg(2+)-ATPase (P < 0.01, P < 0.02). The whole blood viscosity and symptoms showed improvement to an extent and the total effective rate of blood sugar lowering were as follows: fasting blood glucose (FBG) 77.8%, 2hr. postprandial plasma blood glucose (2hr. postprandial PBG)69.4%. Whereas, the placebo group showed no marked improvement in either whole blood viscosity or symptoms and the total effective rate of blood sugar lowering were FBG 41.7%, 2hr. postprandial PBG 38.9%. CONCLUSION: Xianzhen tablet is effective in improving the activities of Na(+)-K(+)-ATPase and Ca(2+)-Mg(2+)-ATPase, as well as the whole blood viscosity, blood sugar lowering and in improving clinical symptoms, it could treat effectively NIDDM patients with deficiency of both Qi and Yin, deficiency of the Kidney and blood stasis.

Adult↗
PubMed1996

[Effect of Paeonia lactiflora on platelet cytosolic free calcium and erythrocyte membrane Ca(2+)-Mg(2+)-ATPase activity in hyperlipid rabbits].

The effect of Paeonia lactiflora (PL) on platelet cytosolic free calcium and erythrocyte membrane Ca(2+)-Mg(2+)-ATPase activity in hyperlipid rabbits were observed. Results showed the level of platelet cytosolic free calcium in the PL group (276.25 +/- 27.00 nmol/L) was significantly lower than that in the cholesterol group (390.88 +/- 70.00 nmol/L), P < 0.01, the basal and calmodulin-stimulated activities of erythocyte membrane Ca(2+)-Mg(2+)-ATP ase in PL group (0.79 +/- 0.05 mumol.pi-1.mg-1.h-1 and 1.34 +/- 0.10 mumol.pi-1.mg-1.h-1) were higher than that in the cholesterol group (0.65 +/- 0.09 mumol.pi-1.mg-1.h-1 and 1.04 +/- 0.13 mumol.pi-1.mg-1.h-1).

Animals↗
PubMed2006

Theoretical study of interaction of urate with li(+), na(+), k(+), be(2+), mg(2+), and ca(2+) metal cations.

The geometries and energetics of complexes of Li(+), Na(+), K(+), Be(2+), Mg(2+), and Ca(2+)metal cations with different possible uric acid anions (urate) were studied. The complexes were optimized at the B3LYP level and the 6-311++G(d,p) basis set. Complexes of urate with Mg(2+), and Ca(2+)metal cations were also optimized at the MP2/6-31+G(d) level. Single point energy calculations were performed at the MP2/6-311++G(d,p) level. The interactions of the metal cations at different nucleophilic sites of various possible urate were considered. It was revealed that metal cations would interact with urate in a bi-coordinate manner. In the gas phase, the most preferred position for the interaction of Li(+), Na(+), and K(+) cations is between the N(3) and O(2) sites, while all divalent cations Be(2+), Mg(2+), and Ca(2+) prefer binding between the N(7) and O(6) sites of the corresponding urate. The influence of aqueous solvent on the relative stability of different complexes has been examined using the Tomasi's polarized continuum model. The basis set superposition error (BSSE) corrected interaction energy was also computed for complexes. The AIM theory has been applied to analyze the properties of the bond critical points (electron densities and their Laplacians) involved in the coordination between urate and the metal cations. It was revealed that aqueous solvation would have significant effect on the relative stability of complexes obtained by the interaction of urate with Mg(2+) and Ca(2+)cations. Consequently, several complexes were found to exist in the water solution. The effect of metal cations on different NH and CO stretching vibrational modes of uric acid has also been discussed.

Algorithms↗
PubMed2001

Sugar complexes with metal(2+) ions: thermodynamic parameters of associations of Ca(2+), Mg(2+) and Zn(2+) with galactaric acid.

A solution study on the complex ability of galactaric acid (GalaH(2)) for complexation with Ca(2+), Mg(2+) and Zn(2+) ions is reported. The stability constants of the complex species are determined by means of potentiometric measurements. From the dependence of stability constants on temperature, the DeltaH degrees and DeltaS degrees values are also determined. The formation of the complex species is an endothermic process and DeltaH degrees and DeltaS degrees values suggest a chelate coordination mode of galactaric acid involving carboxylic oxygen and alpha-hydroxylic group. The prevailing species at acidic or neutral pH is [MGala] (M=Ca(2+), Mg(2+), Zn(2+)) which is also isolated in the solid state and characterised by means of IR spectroscopy. On increasing pH, the [MGalaH(-1)](-) species is also formed, where the coordinated OH group undergoes deprotonation.

Calcium↗
PubMed1996

NMR Characterization of the Na(3)AlP(3)O(9)N and Na(2)Mg(2)P(3)O(9)N Nitridophosphates: Location of the (NaAl)/Mg(2) Substitution.

We report a solid state nuclear magnetic resonance study of (23)Na, (27)Al, and (31)P in two crystalline nitridophosphate phases, Na(3)AlP(3)O(9)N and Na(2)Mg(2)P(3)O(9)N, including two-dimensional multiple-quantum magic angle spinning (MQ-MAS) experiments on (23)Na to separate overlapping lines. The previously described single-crystal structure of Na(3)AlP(3)O(9)N gives crystallographic examples of Al(OP)(6) and P(O[Al,Na])(2)(ONa)(N[P,Na]) environments and three different environments of sodium: two Na(O)(6) and one Na(O)(6)(N). From these observations we characterize the modification of the local environment of phosphorus and show that Mg only substitutes Na in the Na2 site of the Na(2)Mg(2)P(3)O(9)N structure.

Journal Article↗
PubMed1993

Localization of Cys-344 on the (Ca(2+)-Mg(2+)-ATPase of sarcoplasmic reticulum using resonance energy transfer.

4-Bromomethyl-6,7-dimethoxy-coumarin labels the (Ca(2+)-Mg(2+)-ATPase of skeletal muscle sarcoplasmic reticulum at Cys-344. Resonance energy transfer has been used to measure the distance between this site and Lys-515 labelled with fluorescein isothiocyanate as about 37 A. The height of Cys-344 above the phospholipid/water interface has been measured by resonance energy transfer for the ATPase reconstituted into bilayers containing fluorescein-labelled phosphatidylethanolamine; the height was found to be about 45 A. None of these distances was found to alter on changing pH, or on addition of Mg2+, Ca2+ or vanadate. Quenching of the fluorescence of the coumarin-labelled ATPase with KI suggested that the fluorophore is not fully exposed on the ATPase.

Animals↗
PubMed2001

p-Nitrophenylphosphatase activity catalyzed by plasma membrane (Ca(2+) + Mg(2+)ATPase: correlation with structural changes modulated by glycerol and Ca(2+).

The plasma membrane (Ca(2+) + Mg(2+))ATPase hydrolyzes pseudo-substrates such as p-nitrophenylphosphate. Except when calmodulin is present, Ca(2+) ions inhibit the p-nitrophenylphosphatase activity. In this report it is shown that, in the presence of glycerol, Ca(2+) strongly stimulates phosphatase activity in a dose-dependent manner. The glycerol- and Ca(2+)-induced increase in activity is correlated with modifications in the spectral center of mass (average emission wavenumber) of the intrinsic fluorescence of the enzyme. It is concluded that the synergistic effect of glycerol and Ca(2+) is related to opposite long-term hydration effects on the substrate binding domain and the Ca(2+) binding domain.

4-Nitrophenylphosphatase↗
PubMed1999

Intracellular Ca(2+)-Mg(2+)-ATPase regulates calcium influx and acrosomal exocytosis in bull and ram spermatozoa.

Calcium influx is required for the mammalian sperm acrosome reaction (AR), an exocytotic event occurring in the sperm head prior to fertilization. We show here that thapsigargin, a highly specific inhibitor of the microsomal Ca(2+)-Mg(2+)-ATPase (Ca(2+) pump), can initiate acrosomal exocytosis in capacitated bovine and ram spermatozoa. Initiation of acrosomal exocytosis by thapsigargin requires an influx of Ca(2+), since incubation of cells in the absence of added Ca(2+) or in the presence of the calcium channel blocker, La(3+), completely inhibited thapsigargin-induced acrosomal exocytosis. ATP-Dependent calcium accumulation into nonmitochondrial stores was detected in permeabilized sperm in the presence of ATP and mitochondrial uncoupler. This activity was inhibited by thapsigargin. Thapsigargin elevated the intracellular Ca(2+) concentration ([Ca(2+)](i)), and this increase was inhibited when extracellular Ca(2+) was chelated by EGTA, indicating that this rise in Ca(2+) is derived from the external medium. This rise of [Ca(2+)](i) took place first in the head and later in the midpiece of the spermatozoon. However, immunostaining using a polyclonal antibody directed against the purified inositol 1,4,5-tris-phosphate receptor (IP(3)-R) identified specific staining in the acrosome region, in the postacrosome, and along the tail, but not in the midpiece region. No staining in the acrosome region was observed in sperm without acrosome, indicating that the acrosome cap was stained in intact sperm. The presence of IP(3)-R in the anterior acrosomal region as well as the induction, by thapsigargin, of intracellular Ca(2+) elevation in the acrosomal region and acrosomal exocytosis, implicates the acrosome as a potential cellular Ca(2+) store. We suggest here that the cytosolic Ca(2+) is actively transported into the acrosome by an ATP-dependent, thapsigargin-sensitive Ca(2+) pump and that the accumulated Ca(2+) is released from the acrosome via an IP(3)-gated calcium channel. The ability of thapsigargin to increase [Ca(2+)](i) could be due to depletion of Ca(2+) in the acrosome, resulting in the opening of a capacitative calcium entry channel in the plasma membrane. The effect of thapsigargin on elevated [Ca(2+)](i) in capacitated cells was 2-fold higher than that in noncapacitated sperm, suggesting that the intracellular Ca pump is active during capacitation and that this pump may have a role in regulating [Ca(2+)](i) during capacitation and the AR.

Acrosome↗
PubMed1994

Effects of quercetin on Na(+)-K(+)-exchanging ATPase and Ca(2+) Mg(2+)-ATPase in rats.

Quercetin (Que) ig 200 mg.kg-1, qd x 14 d decreased activities of the Na(+)-K(+)-exchanging ATPase (I) of rat brain plasma membranes and heart sarcolemmal and Ca(2+) Mg(2+)-ATPase (II) of heart sarcolemmal membrane. Que 100 mg.kg-1 reduced the activity of I from rat heart sarcolemmal preparation, but had no effect on that from rat brain plasma membranes. The result shows that I of myocardium is more sensitive than that of brain in rat. Que also showed a remarkable inhibitory effect in the II of heart sarcolemma.

Animals↗
PubMed1987

High dose salbutamol in chronic bronchitis: comparison of 400 micrograms, 1 mg, 1.6 mg, 2 mg and placebo delivered by Rotahaler.

Ten patients with chronic bronchitis (Medical Research Council definition) completed a double-blind placebo-controlled study comparing 400 micrograms, 1 mg, 1.6 mg and 2 mg salbutamol powder. Response was measured by spirometry, peak expiratory flow rate, heart rate and tremor at intervals up to 4 hours post-inhalation. Pulmonary function results showed a trend towards higher doses producing improved response and a longer duration of action, with bronchodilatation following 2 mg significantly greater than 400 micrograms. Seven patients developed or had an increase in tremor following a single dose of 2 mg and in one this was considered to be severe. No adverse effects were recorded on ECG.

Administration, Inhalation↗
PubMed1992

Disparate efficacy of tobramycin on Ca(2+)-, Mg(2+)-, and HEPES-treated Pseudomonas aeruginosa biofilms.

Mucoid exopolysaccharide (MEP) obtained from Pseudomonas aeruginosa 579 was suspended in 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) pH 7.2 containing 0.1-10.0 mM of CaCl2.2H2O or MgCl2.4H2O. MEP treated with HEPES or < 5.0 mM of the Ca2+ or Mg2+ salts remained soluble and bound tobramycin in an equilibrium dialysis bioassay. MEP treated with 5.0 or 10.0 mM of the Ca2+ or Mg2+ salts did not bind tobramycin. Five and 10 mM Ca(2+)-treated MEP precipitated but Mg(2+)-treated MEP did not. Pseudomonas aeruginosa 579 biofilms formed using a defined growth medium having < 1 mM Ca2+ or Mg2+ were treated for 1 h with 10 mM HEPES +/- 5.0 mM CaCl2.2H2O or MgCl2.4H2O, prior to an 8-h exposure to HEPES, or the defined growth medium, +/- 125 micrograms/mL of tobramycin. The tobramycin kill kinetics for the HEPES-, Mg(2+)-, and Ca(2+)-treated biofilms were similar and gradual from T = 0-6 h. The viability of the HEPES- and Mg(2+)-treated populations declined sharply (from 6 to 8 h). Bacteria dispersed from the MEP in control biofilms at 0 and 8 h did not grow in the presence of 7.81 micrograms/mL of tobramycin. Thus, binding of tobramycin of P. aeruginosa 579 MEP may not be as influential to the impediment of tobramycin diffusion as is the steric hindrance imposed by the Ca2+ condensation of the polymer.

Binding Sites↗