Giovanni Ronca

Anti-inflammatory activity of chondroitin sulfate

The pharmacokinetics of chondroitin sulfate (CS, Condrosulf, IBSA, Lugano, Switzerland) were investigated in rats and in healthy volunteers using CS tritiated at the reducing end and CS labeled with 131I or 99mTc respectively. A rapid absorption of orally administered CS is observed in rats and in humans when the drug is dissolved in water. Lower and delayed absorption is observed when CS is administered in gastroresistant capsules. The absolute bio-availability is 15 and 12% for rats and humans respectively. The CS shows a tropism for cartilagineous tissues in rats and for knee tissues in humans as demonstrated by scintigraphic analysis with 99mTc-CS. Monomers, oligo and polysaccharides produced by enzymatic hydrolysis of CS appear in the blood and tissues together with native CS. The effects of partially depolymerized (m.m. 3 to 15 kD) and desulfated fractions on human leukocytes were investigated. CS and its fractions inhibit the directional chemotaxis induced by zymosan-activated serum, are able to decrease the phagocytosis and the release of lysozyme induced by zymosan and to protect the plasma membrane from oxygen reactive species. In rats the oral administration of CS significantly decreases granuloma formation due to sponge implants and cell migration and lysosomal enzyme release in carrageenan pleurisy. Compared with nonsteroidal anti-inflammatory drugs (indomethacin, ibuprofen), CS appears to be more effective on cellular events of inflammation than on edema formation. It is noteworthy that CS is devoid of dangerous effects on the stomach, platelets and kidneys. In synovial fluid of patients requiring joint aspiration, treated orally for 10 days with CS (800 mg/day) the hyaluronate concentration and the intrinsic viscosity significantly increased, while collagenolytic activity, phospholipase A2 and N-acetylglucosaminidase (NAG) decreased. These results give an insight into the mechanism of the anti-inflammatory and chondroprotective actions demonstrated by this drug in a number of clinical trials in patients with osteoarthritis.Osteoarthritis is primarily characterized by areas of destruction of articular cartilage and by synovitis. Articular damage and synovitis are secondary to local increase of pro-inflammatory cytokines (interleukin-1beta and tumor necrosis factor-alpha), enzymes with proteolytic activity (matrix metalloproteinases), and enzymes with pro-inflammatory activity (cyclooxygenase-2 and nitric oxide synthase-2). Enhanced expression of these proteins in chondrocytes and in synovial membrane appears associated to the activation and nuclear translocation of nuclear factor-kappaB (NF-kappaB). Chondroitin sulfate (CS) prevents joint space narrowing and reduces joint swelling and effusion. To produce these effects, CS elicits an anti-inflammatory effect at the chondral and synovial levels. CS and its disaccharides reduce NF-kappaB nuclear translocation, probably by diminishing extracellular signal-regulated kinase1/2, p38mitogen-activated protein kinase and c-Jun N-terminal kinase activation. This review discusses the evidence supporting that CS pleiotropic effects in chondrocytes and synoviocytes are primarily due to a common mechanism, e.g., the inhibition of NF-kappaB nuclear translocation.

Postischemic Changes in Cardiac Sarcoplasmic Reticulum Ca2+ Channels : A Possible Mechanism of Ischemic Preconditioning

We investigated the modifications of cardiac ryanodine receptors/sarcoplasmic reticulum Ca2+ release channels occurring in ischemic preconditioning. In an isolated rat heart model, the injury produced by 30 minutes of global ischemia was reduced by preexposure to three 3-minute periods of global ischemia (preconditioning ischemia). The protection was still present 120 minutes after preconditioning ischemia but disappeared after 240 minutes. Three 1-minute periods of global ischemia did not provide any protection. In the crude homogenate obtained from ventricular myocardium, the density of [3H]ryanodine binding sites averaged 372 +/- 18 fmol/mg of protein in the control condition, decreased 5 minutes after preconditioning ischemia (290 +/- 15 fmol/mg, P < .01), was still significantly reduced after 120 minutes (298 +/- 17 fmol/mg, P < .05), and recovered after 240 minutes (341 +/- 21 fmol/mg). Three 1-minute periods of ischemia did not produce any change in ryanodine binding. The Kd for ryanodine (1.5 +/- 0.3 nmol/L) was unchanged in all cases. In parallel experiments, the crude homogenate or a microsomal fraction was passively loaded with 45Ca, and Ca(2+)-induced Ca2+ release was studied by the quick filtration technique. In both preparations, the rate constant of Ca(2+)-induced Ca2+ release decreased 5 and 120 minutes after preconditioning ischemia (homogenate values: 19.7 +/- 1.4 and 18.9 +/- 0.9 s-1 vs a control value of 25.4 +/- 1.7 s-1, P < .05 in both cases) and recovered after 240 minutes (23.0 +/- 1.9 s-1). The Ca2+ dependence of Ca(2+)-induced Ca2+ release was not affected by preconditioning ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Muscle AMP aminohydrolase. III. A comparative study on the regulatory properties of skeletal muscle enzyme from various species

1. 1. The effect of K+, nucleoside triphosphates, ADP and Pi on skeletal muscle AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) from frog, pigeon, guinea pig, rabbit and rat, partially purified by the same method, was investigated. 2. 2. K+ activates the enzyme from all the sources; however, the complete activation of rat and pigeon enzymes is observed at KCl concentrations lower than those required for a complete activation of the enzyme from the other sources. 3. 3. At pH 6.5 and at KCl concentrations higher than 100 mM, ATP, GTP, ITP and Pi inhibit the enzyme from all the species examined; however, at 50–100 mM KCl ATP activates the enzyme from guinea pig and rabbit. At pH 7.1 and 50–200 mM KCl ATP inhibits all the enzymes. 4. 4. AMP deaminase is inhibited to a limited extent by nucleoside triphosphates; the inhibition degree depends on the pH and on the sources; Pi completely inhibits the enzyme. Creatine phosphate also is an inhibitor. Kinetic evidences suggest two distinct sites of binding, one for the nucleoside triphosphate and the other for Pi. 5. 5. ADP at low KCl concentrations strongly activates the enzyme, while at high KCl concentrations the activation is weaker or absent; besides this effect ADP removes the inhibition by nucleoside triphosphates and Pi. 6. 6. The data suggest that the enzyme in resting muscle is partially or completely inhibited; when ADP accumulates in muscle it removes the inhibition by nucleoside triphosphates, Pi and creatine phosphate and the enzyme results activated.

Protection of ischemic rat heart by dantrolene, an antagonist of the sarcoplasmic reticulum calcium release channel.

Abstract Cytosolic Ca2+ overload plays a major role in the development of irreversible injury during myocardial ischemia. Such overload is due at least in part to the release of Ca2+ from the sarcoplasmic reticulum. Therefore, we investigated whether dantrolene, a blocker of the sarcoplasmic reticulum Ca2+ release channel, may protect from ischemic injury. In binding experiments, we determined the effect of dantrolene on [3H]-ryanodine binding in rat cardiac tissue. In perfusion experiments, isolated rat hearts were perfused for 20 min in the working mode, in the presence of 0–45 μM dantrolene. The hearts were then subjected to 30 min of global ischemia and 120 min of retrograde reperfusion. Tissue injury was evaluated on the basis of triphenyltetrazolium chloride (TTC) staining and LDH release. The binding experiments showed that dantrolene displaced 4 nM [3H]-ryanodine with IC50 of 34 μM. In the perfusion experiments, tissue necrosis (i.e., TTC-negative tissue) averaged 28.3±1.6% of the ventricular mass under control conditions. Dantrolene was protective at micromolar concentrations: tissue necrosis decreased to 21.4±1.0% and 8.4±1.4% with 1 μM and 45 μM dantrolene, respectively (P < 0.05 and P < 0.01). Similar results were obtained with regard to LDH release. At low concentrations (up to 4 μM), dantrolene did not produce any significant hemodynamic effect, except for a slight increase in coronary flow, whereas at higher concentration a negative inotropic effect was apparent. In conclusion, dantrolene reduced ischemic injury even at concentrations that did not affect contractile performance. Modulation of sarcoplasmic reticulum Ca2+ release might represent a new cardioprotective strategy.

Isozymes of AMP deaminase in red and white skeletal muscles

A general differentiation in the metabolic pattern of white and red muscles is classically known [l] . Recently differences in these two types of muscles have been observed also at a molecular level with the demonstration of different types of isoenzymes of myosin ATPase [2] , lactate dehydrogenase [3] and phosphorylase kinase [4] . The activity of adenylate deaminase of skeletal muscle is very high in comparison with that of all other tissues, including heart and smooth muscles [5] Our previous distribution data showed that white muscles from different species deaminate AMP four to ten times more effectively than red muscles [6]. These differences can be due either to a different content of a single form of AMP deaminase or to the existence of isoenzymes with distinct kinetic properties, as observed for the above mentioned key regulatory enzymes. The results of this paper give strong evidence that different types of adenylate deaminase exist in white and red muscles and we demonstrate that, besides quantitative, also qualitative dissimilarities exist.

Protection of isolated rat heart from oxidative stress by exogenous creatine phosphate

The influence of exogenous creatine phosphate (CP) on peroxidative heart injury was investigated in two experimental models: isolated working rat hearts and myocardial membrane preparations. In the first model the addition of 190 microM hydrogen peroxide to the perfusion buffer caused a marked decrease of aortic flow, minute work and peak aortic pressure, and leakage of intracellular enzymes. In the presence of 10 mM CP the hemodynamic damage produced by the same concentration of hydrogen peroxide was significantly lower and enzyme release was also remarkably reduced. The protection was concentration-dependent and the whole structure of the molecule was required since creatine was found to be ineffective. In the absence of hydrogen peroxide, CP and creatine did not affect heart performance. In microsomal membrane preparations CP decreased the formation of thiobarbituric acid-reactive material (malonaldehyde) induced by hydrogen peroxide in the presence of ferrous ions. This protection was concentration-dependent and occurred at physiological concentrations of CP. Also in this experimental model creatine had no effect and creatine plus inorganic phosphate was much less active than CP. The influence of CP on oxidative heart stress could account for the beneficial effect of this substance in different models of ischemic injury.

Reversing of chlorambucil resistance by ethacrynic acid in a B-CLL patient

Summary We evaluated the reversing activity of ethacrynic acid in a B‐CLL patient resistant to chlorambucil. The glutathione S‐transferase (GST) activity, measured in peripheral blood lymphocytes, resulted extremely elevated. Etha crynic acid, at pharmacological concentrations, partially reversed chlorambucil resistance and this result appeared related to the increased GST levels.

A novel mutation in the pendrin gene associated with Pendred's syndrome

Pendreds syndrome is an autosomal recessive disorder characterized by goitre, sensorineural deafness and iodide organification defect. It is one of the most frequent causes of congenital deafness, accounting for about 10% of hereditary hearing loss. It is caused by mutations in the pendrin (PDS) gene, a 21 exon gene located on chromosome 7. The aim of this study was to examine an Italian family affected with Pendreds syndrome at the molecular level.

Interaction of rat muscle AMP aminohydrolase with chelating agents and metal ions.

AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) from rat skeletal muscle behaves as a regulated enzyme. It is activated by some monovalent cations, the cationactivated enzyme is inhibited by nucleoside triphosphates, creatine phosphate and phosphate; ADP removes the inhibition by these compounds [I] . Some organic anions as lactate, acetate and citrate are also inhibitors. In the presence of citrate the enzyme becomes insensible to the nucleoside triphosphate inhibition [2]. Recently Zielke and Suelter [3,4] reported that rabbit AMP deaminase which showed similar kinetic and regulatory properties to the rat enzyme [ 1,2,5,6] was a metal10 protein containing Zn2+. This paper gives evidence that Zn2+ is a firmly bound component (2 atoms per mole of enzyme) of the rat AMP deaminase and is essential for the enzyme activity. The metal ion can be dissociated from the protein by incubation with chelating agents; some enzyme effectors affect the rate of inactivation by chelating compounds. The inactive apoenzyme can be reactivated by the addition of Zn2+. Some other bivalent metal ions can also partially reactive AMP deaminase.

Effect of cardiac A1 adenosine receptor overexpression on sarcoplasmic reticulum function

OBJECTIVE We investigated the effect of A(1) adenosine receptor overexpression, which has been reported to increase myocardial tolerance to ischemia-reperfusion injury, on sarcoplasmic reticulum (SR) Ca(2+) handling. METHODS Transgenic mouse hearts (approximately 300-fold A(1) adenosine receptor overexpression) and wild-type mouse hearts were perfused in the Langendorff mode and subjected either to 80 min of aerobic perfusion or to 30 min of aerobic perfusion, 20 min of global ischemia and 30 min of reperfusion. The hearts were then homogenized and used to assay SR oxalate-supported 45Ca(2+) uptake and [3H]-ryanodine binding. RESULTS Transgenic hearts showed increased resistance to ischemia-reperfusion, as shown by lower diastolic tension (1.5 +/- 0.2 vs. 2.6 +/- 0.1 g, P<0.05) and higher recovery of developed tension (45 +/- 3 vs. 30 +/- 4% of the baseline, P<0.05) following ischemia-reperfusion. Under baseline conditions, oxalate-supported 45Ca(2+) uptake was lower in transgenic hearts, owing to reduced V(max) (10.6 +/- 2.0 vs. 17.8 +/- 2.7 nmol/min per mg of protein, P<0.05), and the difference was preserved after ischemia-reperfusion (10.0 +/- 1.0 vs. 15.7 +/- 2.5 nmol/min per mg of protein, P<0.05). No significant difference in [3H]-ryanodine binding was observed. CONCLUSIONS A(1) adenosine receptor overexpression is associated with a decreased rate of active Ca(2+) transport into the SR. We hypothesize that changes in SR function may cause a depletion of the SR Ca(2+) pool, which might protect from ischemic injury by delaying the development of cytosolic Ca(2+) overload during ischemia.