Amorphous Calcium Phosphate-Mediated Binding of Matrix Metalloproteinase-9 to Fibrin is Inhibited by Pyrophosphate and Bisphosphonate

Coordinate regulation of fibrinolytic and collagenolytic systems is essential for normal tissue remodeling and wound healing. To define the molecular mechanisms which link these two proteolytic systems, we have investigated the role of fibrin in matrix metalloproteinase (MMP) function. Both active and latent forms of MMP-9 (gelatinase B) bind to fibrin in a selective, dose-dependent manner; latent enzyme is activated by plasmin during fibrinolysis. Fibrin binding of MMP-9 is mediated by amorphous calcium phosphate (ACP), and proceeds in a step-wise fashion with formation of ACP as the first and rate-limiting step. MMP-9 rapidly binds preformed ACP to yield a transient ACP: MMP-9 complex that avidly binds fibrin. Here we report the effect(s) on fibrin: ACP: MMP-9 formation/dissociation of pyrophosphate (POP), an endogenous calcification inhibitor, and its bisphosphonate analog, alendronate (PCP). MMP-9 was obtained from neutrophil lysate and ACP formation was monitored turbidimetrically. Free MMP-9, ACP: MMP-9 and fibrin: ACP: MMP-9 complexes were analyzed by gelatin zymography. POP at physiologic concentrations (0.5-2.5 microM) inhibited both ACP formation and subsequent fibrin binding of MMP-9 at orthophosphate concentrations of 250 microM. PCP exhibited a similar inhibitory effect. With both substances, inhibition was slightly overcome (>2.5 microM) by higher phosphate (500 microM). In contrast, supraphysiologic concentrations of either POP or PCP (>50 microM) were required to inhibit MMP-9 binding to preformed ACP or to induce dissociation of preformed ACP: MMP-9 complexes (50-100 microM). Neither POP nor PCP had any effect on preformed fibrin: ACP: MMP-9 at concentrations up 1 mM. POP is an endogenous by-product of numerous metabolic pathways and may regulate bone turnover, soft tissue calcification, and contribute to the pathogenesis of calcium pyrophosphate crystal disease (CPPD). These studies support another role for POP and fibrin: ACP: MMP-9 complexes in physiologic and pathologic processes, including tumorigenesis and cancer metastasis.     

Interaction of Amorphous Calcium Phosphate with Fibrin In Vitro Causes Decreased Fibrinolysis and Altered Protease Profiles: Implications for Atherosclerotic Disease

Previously, we demonstrated that amorphous calcium phosphate (ACP), chemical precursor to apatite, strongly interacted with fibrin and facilitated binding of matrix metalloproteinase (MMP)-9, a type IV collagenase. Plasmin-dependent fibrinolysis resulted in coordinate MMP-9 activation. Here we report on the effect(s) of ACP on fibrin degradation and binding of endogenous plasma proteases. Electrophoresis (8.5% SDS-PAGE) revealed that fibrin formed in the presence of ACP demonstrated characteristic - dimers (90-kDa) and -monomers (55-kDa), but resisted spontaneous fibrinolysis (72 h, 37°C) or degradation by plasminogen activators (uPA, tPA). Casein zymography revealed an ACP-dependent decrease in fibrin binding of a low molecular weight (Mw) protease triplet (47-, 43-, 42-kDa) and increased fibrin binding of two high Mw proteases (94- and 84-kDa). The low Mw triplet also possessed gelatinolytic activity, but was not an MMP since 1,10-phenanthroline was ineffective as an inhibitor. Fibrin-binding proteases were inhibited to some degree by the serine protease inhibitor aprotinin. Competition/dissociation experiments with -aminocaproic acid revealed that the low Mw triplet lacked kringle regions whereas the 94- and 84-kDa proteases were tentatively identified and glu-/lys-plasmin(ogen)s. The triplet may, however, represent one or more kringle deficient mini-plasminogen(s), since electrophoretic mobility and substrate specificity was similar to elastase-generated mini-plasminogen. To explore these findings in a clinically relevant setting, a series of plasma samples was collected from a patient with unstable angina prior to, during, and post coronary artery bypass graft (CABG) surgery. Fibrin formed from plasma collected during and immediately post CABG was associated with increased fibrinolytic capacity and enhanced binding of a) MMP-9, b) the low Mw protease triplet (described above), and c) PA (as putative 110-kDa tPA:PAI-1 complex). The relevance of these findings to pathologic calcification of atherosclerotic plaques is discussed.     

Binding of Latent Matrix Metalloproteinase 9 to Fibrin: Activation via a Plasmin-Dependent Pathway

The binding of two matrix metalloproteinases (MMP) to fibrin was evaluated. MMP-2 (72-kDa) and MMP-9 (92-, 130-, and 225-kDa) were selected since both contain a fibronectin-like region and fibronectin binds fibrin. Gelatin zymography indicated selective and dose dependent binding of MMP-9 to fibrin. No MMP-2 binding to fibrin occurred. Densitometry revealed that the 130- and 225-kDa forms demonstrated similar sigmoidal binding profiles whereas 92-kDa uptake was hyperbolic. Fibronectin and TIMP-1 competition studies indicated that the fibronectin and C-terminal MMP-9 domains, respectively, were not involved with fibrin binding. The MMP-9 collagen-like region may be of regulatory significance since type I and II fibrillar and type IV basement membrane collagens demonstrated fibrin binding. During fibrinolysis, latent fibrin-bound MMP-9 was processed to lower molecular weight forms consistent with proteolytic activation. This process was inhibited by -aminocaproic acid, indicating a plasmin-dependent pathway. The significance of these findings to procoagulant activity and MMP-mediated extracellular matrix destruction during inflammation and tumor invasion and metastasis is discussed.     

Replenishment of the cellular calcium required for non-immunologic stimulation of mast cell histamine secretion: Temperature sensitivity and inhibition by manganese and sodium-free conditions

Mast cells depleted of cellular calcium (Ca) by a 3 hr exposure to Ca-free conditions and then bathed in Ca-free Locke failed to release histamine when stimulated by compound 48/80 or peptides. The cellular Ca required for histamine release could be replenished by a 5 sec exposure to extracellular Ca at 37°C. To inhibit this replenished cellular Ca dependent histamine secretion required an additional 3 hr exposure to Ca-free conditions. When cellular Ca was replenished at 4°C, an additional 2 min incubation at 37°C was required to restore stimulated secretion to a maximum. During this 2 min incubation period the replenished cellular Ca is suggested to be processed so that it can be used for secretion. Manganese (Mn) or cobalt added during (but not after) this 2 min incubation period prevented the restoration of histamine release. Preincubation of cellular Ca depleted mast cells in Mn (0.1–1 mM) blocked the effect of subsequent Ca replenishment at 37°C while coblat and barium were less inhibitory. Neither magnesium nor strontium were inhibitory. Extracellular sodium (Na) was required for the restoration of cellular Ca dependent histamine secretion. Lithium could substitute for Na but rubidium and potassium were ineffective.Supported by grants AI 19436 and AI 19736 from the National Institutes of Health.     

Bradykinin Stimulates MMP-2 Production in Guinea Pig Tracheal Smooth Muscle Cells

The implication of bradykinin (BK) receptors in the release of the matrix metalloproteinase-2 (MMP-2; gelatinase A) was studied in guinea pig tracheal smooth muscle cells (GP-TSMC). Bradykinin (10^–8–10^–4 M) induced a time- and concentration-dependent upregulation of MMP-2 production from cultured GP-TSMC. Pretreatment of the GP-TSMC with the bradykinin B₂ receptor (BKB-R) antagonist Hpp-HOE-140 (Hpp-D-Arg⁰-Hyp³-Thi⁵-D-Tic⁷-Oic⁸-BK; 10^–8–10^–4 M) significantly inhibited the BK-stimulated upregulation of MMP-2 in GP-TSMC in a concentration-related manner. Conversely, GP-TSMC pretreated with the selective bradykinin B₁ receptor (BKB₁-R) antagonist R-954 (Ac-Orn[Oic², -MePhe⁵, D-Nal⁷, Ile⁸]desArg⁹BK; 10^–8–10^–4 M) did not show any change in the response to BK. Moreover, the selective BKB₂-R agonist Lys⁰BK (kallidin; 10^–8–10^–4 M) stimulated whereas the selective BKB₁-R agonist desArg⁹BK (DBK; 10^–8–10^–4 M) had no effect on MMP-2 release from GP-TSMC. Further, the nonselective cyclooxygenase (COX) enzyme inhibitor indomethacin (IND; 10^–5 M), the glucocorticosteroid dexamethasone (DEX; 1 ng/mL) and the protein synthesis inhibitors, cycloheximide (CHX; 10^–6 M) and actinomycin D (ACT-D; 10^–8 M) also inhibited BK-induced MMP-2 release from GP-TSMC. These results provide the first evidence for the involvement of BK in the release of MMP-2 from airway smooth muscle cells through activation of the BKB₂-R. Such response is mostly mediated by the induction of COX and the subsequent production of endogenous prostaglandins (PGs). It could therefore be suggested that MMP-2 might play a role in the process of airway remodeling.     

Effects of neomycin on calcium channel currents in clonal GH3 pituitary cells

Membrane Ca currents were recorded from voltage-clamped clonal (GH3) pituitary cells under conditions where currents through Na and K channels were abolished. Two Ca currents, with distinct kinetics and voltage dependence for activation and inactivation, were identified. Neomycin, an aminoglycoside polycation, inhibited both the transient (I _Ca,t) and the slowly-inactivating (I _Ca,s) Ca currents in a dose-dependent manner (100–1,000 M). The blockade was reversible andI _Ca,s was more sensitive to neomycin thanI _Ca,t. The inhibition ofI _Ca,s was frequency and time-independent, and was not affected by changes in the holding membrane potential (–35 to –100 mV). Neomycin did not affect the voltage dependence for inactivation ofI _Ca,t. The blockade of both Ca currents by neomycin is ascribed to the general property of aminoglycosides to compete with, and displace Ca ions from membrane binding sites that determine the currents and selectivity of Ca channels. Because comparable concentrations of neomycin were required for blocking the currents conveyed by Ca or, in the absence of external divalent cations, by Na ions through the slowly-inactivating Ca channels, we suggest that the neomycin binding sites are distinct from the high-affinity transition sites within the Ca channel path.     

Bradykinin induces a B2 receptor-mediated calcium signal linked to prostanoid formation in human gingival fibroblastsin vitro

The aim of the study was to determine the effect of bradykinin (BK) on the level of cytoplasmic-free Ca²⁺, [Ca²⁺]_i, in human gingival fibroblasts and its relation to BK-induced prostanoid formation. BK, but not des-Arg⁹-BK, induced a significant rapid (within seconds) and transient increase in [Ca²⁺]_i, that was not dependent on extracellular Ca²⁺. The stimulatory effect of BK was seen in concentrations at or above 10^–8 M, with the most pronounced effect at 10^–6 M.d-Arg⁰–Hyp³–Thi^5,8–dPhe⁷-BK, a BK B2 receptor antagonist, but not des-Arg⁹–Leu⁸-BK, a BK B1 receptor antagonist, blocked BK-induced rise in [Ca²⁺]_i. The BK B2 receptor antagonist also significantly reduced BK-induced PGE₂ formation. When extracellular Ca²⁺ in the incubation medium was depleted, either by addition of EGTA or by omission of Ca²⁺ addition, BK still caused a significant stimulation of PGE₂ formation. The calcium ionophores A23187 and ionomycin, similar to BK, caused a burst of PGE₂ formation. The two phorbol esters phorbol 12,13-dibutyrate and 4--phorbol-didecanoate positively amplified calcium ionophore A23187-induced PGE₂ formation. The results indicate that BK-induced PGE₂ formation in gingival fibroblasts is coupled to an increase in [Ca²⁺]_i mediated by the BK B2 receptor, and which is independent of extracellular Ca²⁺.     

Currents carried by sodium ions through transient calcium channels in clonal GH3 pituitary cells

Summary The transient or T-type Ca channel currents of voltage-clamped clonal (GH₃) pituitary cells were studied after blockade of Na and K channels. Removal of Ca ions from the bathing medium abolished the Ca currents and allowed Na ions to convey current through the transient channels. These Na currents (I_Na,t) were activated at threshold potentials of ca. –85 mV, reached peak amplitudes (up to 2,000 pA) at –60 mV, and were 50% inactivated at holding membrane potentials of –110 to –120 mV. The time constant for inactivation of the maximal I_Na,t was 12±2.4 msec (compared with 22.1±0.85 msec for maximal I_{Ca, t}). The I_Na,t was insensitive to tetrodotoxin (1 M) and to nimodipine (200 nM) but was abolished by 100 M external [Ca²⁺]. It is suggested that high affinity Ca binding sites in the channel modulate the ion selectivity and the current flow through T-type Ca channels.     

Receptors for neurohypophyseal hormones along the rat nephron: 125I-labelled d(CH2)5[Tyr(Me)2, Thr4, Orn8, Tyr-NH 2 9 ] vasotocin binding in microdissected tubules

A microassay was developed to measure the binding of the labelled monoiodinated analogue [1-(mercapto-,-cyclopentamethylenepropionic acid), 2-O-mithyltyrosine, 4-threonine, 8-ornithine, 9-¹²⁵I-tyrosylamide]vasotocin ¹²⁵I-d(CH₂)₅[Tyr (Me)², Thr⁴, Tyr-NH ₂ ⁹ ]OVT to isolated nephron segments microdissected from collagenase-treated rat kidneys. When determined using 1.7 nM labelled ligand at 4° C, specific binding sites (expressed at 10^–18 mol ¹²⁵I-d(CH₂)₅[Tyr (Me)², Thr⁴, Tyr-NH ₂ ⁹ ]OVT bound/mm tubule length) were found in medullary thick ascending limbs (MTAL), 1.67±0.49; cortical thick ascending limbs, 2.20±0.80; cortical collecting ducts, 2.39±0.86; outer medullary collecting ducts (OMCD), 2.54±0.53 and inner medullary collecting ducts, 5.33±0.40, whereas no specific binding could be detected in glomeruli and proximal tubules. Specific ¹²⁵I-d(CH₂)₅[Tyr (Me)², Thr⁴, Tyr-NH ₂ ⁹ ]OVT binding to OMCD was saturable with incubation time and reversible after elimination of free labelled ligand (the association and dissociation rate constants at 4° C were 1.06×10⁷ M^–1 min^–1 and 1.95×10^–2 min^–1 respectively). The stereospecificity of MTAL and OMCD binding sites was assessed in competitive experiments revealing the following recognition pattern for a series of eight vasopressin analogues:ddAVP>AVP>d(CH₂)₅-[Tyr (Me)², Thr⁴, Tyr-NH ₂ ⁹ ]OVT=AVT=OT>d(CH₂)₅[Tyr(Me)²]AVP=[Thr⁴, Gly⁷]OT>[Phe², Orn⁸]VT, whereas pharmacological concentrations of insulin and glucagon did not impair radioligand binding. These results indicate that the detected labelled binding sites might correspond mainly to physiological V₂ vasopressin receptors.     

Voltage-activated calcium channels in rat Purkinje cells maintained in culture

Cell-attached patch recordings were used to study calcium channels on the dendritic membrane of rat cerebellar Purkinje cells maintained in culture. Experiments were performed with isotonic BaCl₂ (110 mM) in the pipette and isotonic potassium gluconate in the bath to zero the cell membrane potential. Two distinct types of voltage-activated calcium channels were identified. The first one had a small conductance (9 pS), was activated at a low threshold ( –50 mV) and could be inactivated by holding the membrane potential at –30 mV. This channel had the same characteristics as the T channel described in other neuronal preparations. The second type of Ca channel activated at a high threshold (–30 or +10 mV depending on whether BAY K 8644 was added or not to the pipette solution) and was still activatable even when the membrane was held at –40 mV. In the presence of BAY K 8644 this channel had a conductance of 21 pS with long openings. All these characteristics are similar to those of the S (L) Ca channel described in many preparations. The present study is in agreement with our previous experiments on Purkinje dendrites, where we identified low and high threshold Ca currents using the whole-cell configuration. Up to now, no channel corresponding to the N current has been observed but we cannot exclude us presence.Present Andress: Centre CNRS-INSERM de Pharmacologie-Endocrinologie, rue de la Cardonille, B.P. 5055, 34033 Montpellier, France     

Relaxation rate of intact striated muscle fibres after flash photolysis of a caged calcium chelator (diazo-2)

Summary Single twitch fibres from lumbrical muscles ofXenopus have been loaded with the photolysable calcium-chelator diazo-2 by incubation in Ringer solution containing the membrane permeable acetoxymethyl ester (AM) form of diazo-2. Incubation caused a progressive slowing of tetanus rise and relaxation which is ascribed to calcium-buffering by unphotolyzed diazo-2 (K_d=2.2 M). After incubation, exposure to a brief UV flash caused a three to four fold increase in the rate of tension fall. A flash given 16–18 ms after the last tetanic stimulus (at 22–24°C) resulted in 10% increase in relaxation rate compared with the control before incubation. A much bigger effect was observed when a flash was given half-way into the slow phase, where an 1.8–1.9-fold increase in relaxation rate, above the preincubation slope, was observed. It is concluded that rapid lowering of [Ca]_i, and hence more rapid removal of Ca²⁺ from troponin, speeds up relaxation, indicating that calcium translocation is the major determinant of the rate of tension fall during the isometric phase of relaxation.     

A human myeloma immunoglobulin G binding four moles of calcium associated with asymptomatic hypercalcemia

A calcium-binding immunoglobulin G (IgG_I^RUP) was identified in the serum of a patient with multiple myeloma, asymptomatic hypercalcemia, and a normal ionized serum calcium. Calcium binding by IgG^RUP was confirmed by two-dimensional electrophoresis with calcium-45 and equilibrium dialysis. Amino acid analyses indicated an unusually high number of glutamic (or glutamine) residues in the L chain and Fab fragment but no detectable -carboxyglutamic acid. As determined by equilibrium dialysis with⁴⁵Ca, the intact IgG^RUP and its Fab fragments bound calcium at an optimum pH of 7.4. There was minimal binding of calcium to H chains and no binding by L chains or the Fc fragment. Recombination of H and L chains partially restored the binding activity. By Scatchard analysis, the binding affinity (K _d) of IgG^RUP was 1.7×10^–3 M and the binding capacity was 4 mol of calcium/mol of IgG. The binding of 4 mol of calcium/mol of IgG is twice that reported previously for two other calcium-binding myeloma proteins and suggests unique properties of IgG^RUP.     

Forearm temperature profile during the transient phase of thermal stress

Summary The transient temperature response of the resting human forearm immersed in water at temperatures (T _w) ranging from 15 to 36°C was investigated. Tissue temperature (T _t) was continuously monitored by a calibrated multicouple probe during the 3-h immersions.T _t was measured every 5 mm, from the longitudinal axis of the forearm to the skin surface. Skin temperature, rectal temperature, and blood flow ( ) were also measured during the immersions. The maximum rate of change of the forearm mean tissue temperature ( ) occurred during the first 5 min of the immersion. was linearly dependent onT _w (P<0.001), with mean values (SEM) ranging from –0.8 (0.1) °C · min^–1 at 15°C to 0.2 (0.1) °C · min^–1 at 36°C. The maximum rate of change of compartment mean temperature was dependent (P<0.001) on the radial distance from the longitudinal axis of the forearm. The half-time for thermal steady state of the forearm mean tissue temperature was linearly dependent onT _w between 30 and 36°C (P<0.01), with mean values (SEM) ranging from 15.6 (0.6) min at 30°C to 9.7 (1.2) min at 36°C and not different between 15 and 30°C, averaging 16.2 (0.6) min. There was a significant linear relationship between the half-time for thermal steady-state of the compartment mean temperature and the radial distance from the longitudinal axis of the forearm for each value ofT _w tested (P<0.001). The data of the present study suggest that the forearm is an important determinant of the transient thermal response of the forearm tissue during thermal stress.     

Exercise thermoregulation after 6 h of chair rest, 6° head-down bed-rest,and water immersion deconditioning in men

The purpose was to investigate the mechanism for the excessive exercise hyperthermia following deconditioning (reduction of physical fitness). Rectal (T _re) and mean skin ( ) temperatures and thermoregulatory responses were measured in six men [mean (SD) age, 32 (6) years; mass, 78.26 (5.80) kg; surface area, 1.95 (0.11)m²; maximum oxygen uptake ( ), 48 (6) ml·min^–1·kg^–1; whilst supine in air at dry bulb temperature 23.2 (0.6)°C, relative humidity 31.1 (11.1)% and air speed 5.6 (0.1) m·min^–1] during 70 min of leg cycle exercise [51 (4)% ] in ambulatory control (AC), or following 6 h of chair rest (CR), 6° head-down bed rest (BR), and 20° (WI20) and 80° (WI80) foot-down water immersion [water temperature, 35.0 (0.1)°C]. Compared with the AC exercise T _re [mean (SD) 0.77 (0.13)°C], T _re after CR was 0.83 (0.08)°C (NS), after BR 0.92 (0.13)°C (^*P<0.05), after WI80 0.96 (0.13)°C^*, and after WI20 1.03 (0.09)°C^*. All responded similarly to exercise: they decreased (NS) by 0.5–0.7°C in minutes 4–8 and equilibrated at +0.1 to +0.5°C at 60–70. Skin heat conductance was not different among the five conditions (range = 147–159 kJ·m^–2·h^–1·°C^–1). Results from an intercorrelation matrix suggested that total body sweat rate was more closely related toT _re at 70 min (T _re70) than limb sweat rate or blood flow. Only 36% of the variability inT _re70 could be accounted for by total sweating, and less than 10% from total body dehydration. It would appear that multiple factors are involved which may include change in sensitivity of thermo- and osmoreceptors.     

Digital-imaging microscopy analysis of calcium release from sarcoplasmic reticulum in single rat cardiac myocytes

Digital imaging microscopy of fura-2 fluorescence has allowed us to assess the dynamic patterns of local Ca increase in newly isolated rat myocardial cells. Of the myocytes bathed in a saline solution (1.8 mM Ca²⁺, 37°C, pH 7.4), 10%–20% exhibited local spontaneous contractions. The resting intracellular free calcium concentration ([Ca²⁺]_i) of these cells was 106±4nM versus 77±3 nM for non-contracting cells. The spontaneous contractile activity appeared to be closely related to internal spontaneous Ca waves that spread across the myoplasm (velocity 50 m/s, maximal Ca amplitude=195±11nM) along the major axis of the cells. Precise topographical examination of Ca wave propagation indicated a refractory period for internal Ca release. The occurrence of both the generation and propagation of spontaneous Ca increases appeared to be closely dependent on the extent of Ca loading of the cells. Most of our observations were in accordance with the assumption that local Ca overload of the sarcoplasmic reticulum (SR) is the main parameter involved in the spontaneous Ca-release phenomena. Using the same approach, the increase in internal Ca evoked by KCl (50 mM) addition was investigated, and compared with that seen during spontaneous activity. Total [Ca²⁺]_i increase induced by K⁺ depolarization involved three consecutive local Ca-release patterns: (a) a peripheral Ca enhancement that remained during the total [Ca²⁺]_i increase, (b) subsequent transversal local Ca increases occurring in Z-line regions, (c) longitudinal local Ca increases. In addition, a weak heterogeneous Ca distribution was detected in both peripheral and central parts of resting cardiac cells. Thus, the total Ca increase seemed to result consecutively from a peripheral Ca pool, from junctional SR and from longitudinal structures (possibly longitudinal SR).     

Ion permeation through single ACh-activated channels in denervated adult toad sartorius skeletal muscle fibres: effect of temperature

The gigaohm seal technique was used to study the effects of temperature on ion permeation through acetylcholine-activated channels. This was done in cell-attached patches of the extrajunctional membrane of chronicallydenervated, enzyme-treated cells from sartorius muscle of the toadBufo marinus. The predominant extracellular cation in the pipette solution was Na⁺. Single channel currentvoltage curves were measured at different temperatures and electrodiffusion and three-site-four-barrier rate theory models were used to characterize ion permeation through the channels and determine the effects of temperature on permeation parameters. The fitting of the experimental data to these models suggested the presence of at least three and probably more ion-selective sites within the channel. The most frequently occuring channel type (>95% of channel openings) had a chord conductance of 25 pS at 11°C and –70 mV and was classified as extrajunctional. The single channel conductance of this channel had a low temperature-dependence (Q ₁₀1.3). The apparent activation enthalpy, E_a, for the conductance between 11°C and 20°C, did not appear to be significantly voltage-sensitive and had a value of about 17±2 kJ·mol^–1 at a voltage of –70 mV. The Arrhenius plot of conductance appeared linear between 11 and 20°C at all potentials examined. The data was consistent with a break in the slope of the Arrhenius plot at temperatures between 5 and 11°C at all potentials examined, suggesting a possible phase transition of the membrane lipids. In contrast to the relative permeability, which was not very temperature sensitive, the relative binding constant was significantly affected by temperature. The relative Na/K binding constant sequence was:K _5°C> K _20°C> K _15°CK _11°C. In addition, the decrease in conductance observed at the most depolarized potentials was accentuated as the temperature was increased, suggesting a rate-limiting access step for ions from the intracellular solution into the channel.     

Über die unterschiedliche Wirkung von Calcium und Noradrenalin auf den Sauerstoffverbrauch isolierter Meerschweinchenvorhöfe

Zusammenfassung An isolierten, elektrisch gereizten (240/min, 5 msec, 20 V) inken Meerrschweinchenvorhöfen wurde mit einer früher beschriebenen Methode (Klaus et al., 1968a), die eine gleichzeitige und fortlaufende Bestimmung des O₂-Verbrauches (polarographisch) und der Kontraktionskraft (isometrisch) erlaubt, die Wirkung verschiedener Calcium-(Ca 0,9; 1,8; 3,6; 7,2 mM/l) und Noradrenalin-(NA 10^–11; 10^–9; 10^–7; 10^–5 g/ml) Konzentrationen untersucht (Tyrodelösung, 37°C).Der Ruhe-O₂-Verbrauch der mit 1 g vorbelasteten Vorhöfe betrug 0,194 . Er wurde durch keine der untersuchten Ca- und NA-Konzentrationen signifikant beeinflußt.Reizung erhöhte den O₂-Verbrauch (bei 0,9 mM/l Ca) um . Die Kontraktionskraft stieg unter steigenden Ca- und Noradrenalin-Konzentrationen von ca. Der O₂-Verbrauch wurde durch Ca konzentrationsabhängig gesteigert. Unter NA 10^–11 g/ml fand sich eine statistisch signifikante (p<0,01) Senkung des O₂-Verbrauches. Bei 10^–9 und 10^–7 g/ml NA war er nicht vom Kontrollwert unter-schieden und nahm erst bei 10^–5 g/ml sehr stark zu.Der Wirkungsgrad wurde durch Ca zwischen 0,9 und 1,8 mM/l um ca. 50% erhöht und erreichte damit sein Maximum.Noradrenalin in Konzentrationen von 10^–11 und 10^–9 g/ml steigerte den Wirkungsgrad um ca. 150%. Weitere Erhöhung der NA-Konzentration bewirkte einen steilen Abfall des Wirkungsgrades, der aber auch bei 10^–5 g/ml noch um ca. 25% gegenüber den Kontrollen erhöht war.Der Vergleich der Wirkungen von Ca und Noradrenalin zeigt, daß für gleiche Kontraktionskraftsteigerung der O₂-Verbrauch unter Noradrenalin immer geringer war als er unter Ca gefunden wurde. Die Befunde werden über die positiv bathmotrope Wirkung des Noradrenalin sowie über Veränderungen im Ca-Umsatz der Herzmuskelzelle und deren Einfluß auf den oxydativen Stoffwechsel erklärt.     

Quantitative localization of Na-K pump sites in the frog sacculus

Summary The Na-K pump site distribution within the macula, perimacula, and wall epithelia of the sacculus in the frog inner ear was examined with quantitative [³H]ouabain autoradiography. Excised tissue was incubated for 10–30 min (23 ° C) in micromolar concentrations of high specific activity [³H]ouabain (14–70 Ci mT^–1, 5–15 Ci mmor^–1), washed for 30 min (4 ° C), then rapidly frozen (–175 ° C) and processed for light and electron microscope autoradiography. Control experiments based on (1) high K⁺ (50 mm) in the incubation and (2) low specific activity [³H]Jouabain (1 mM, 0.013–0.025 Ci mmol^–1) indicated negligible nonspecific binding of the [³H]ouabain.Measurable levels of specific [³H]ouabain binding occurred in all saccular regions examined. Binding was localized to the basolateral cell membranes with no detectable binding to the apical membranes. [³H]ouabain binding across the apical-basal axis of the saccule macular epithelium was nonuniform. Binding was low in the apical region, rose to a peak in the middle two-thirds, and then fell again close to the basement membrane. Electron microscope autoradiography suggested that this peak was due to ouabain binding to nerve terminals. Denervation of the sacculus eliminated the peak in [³H]ouabain binding and quantitative grain density analysis revealed that 45% of the Na-K pumps within the saccule macula were located on the nerve terminals.Na-K pump site density per unit volume was estimated by quantitative grain density analysis and the following values were obtained (sites m^–3 × 10³, means ± S.E.M.): saccule macula, 1.9 ± 0.2; saccule perimacula, 1.1 ± 0.1; saccule wall, 2.3 ± 0.3. Stereological analysis of conventionally fixed tissue was used to estimate overall plasma membrane surface area per unit volume (S _v). Na-K pump site densities per unit membrane area for the various regions were calculated by combining the autoradiographical and Stereological data. The following values were obtained (sites m^–2 ± 25%): saccule macula, 2500; saccule perimacula, 2500. Values for individual cells within the macula (sites m^–2 ± 25%) were: hair cells, 3000; nerve terminals, 3000; supporting cells, 1500.     

Effects of calcium channel blockers on the contractility of the filariidAcanthocheilonema viteae

The role of calcium in muscle contractility was explored in the filarial nematodeAcanthocheilonema viteae (Dipetalonema viteae). The parasite was slit open longitudinally and mounted in a smooth-muscle chamber that had been filled with aerated (95% N₂/5% CO₂) physiological solution at 37°C. Nifedipine (10^–6 m) and cadmium (3×10^–5 m) reduced the spontaneous isotonic contractions ofA. viteae, whereas verapamil (10^–5 m) and diltiazem (10^–5 m) enhanced them. The effects of nifedipine and verapamil did not appear to be due to the solvent ethanol. All of the drugs reduced the maximal contraction induced by acetylcholine (ACh, 10^–5 m), although nifedipine was the most potent. After the exposure of worm preparations to a calcium-free medium containing ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA, 10^–4 m) for 1 h, application of ACh (10^–5 m) induced a small, transient contraction. Subsequent applications of ACh in this medium had no effect. Thus, the nematode muscle contraction appears to depend on extracellular calcium. Nifedipine, diltiazem, and verapamil could act by reducing the calcium influx across the muscle membrane.     

Matrix metalloproteinases in type 2 diabetes and non-diabetic controls: effects of short-term and chronic hyperglycaemia

Introduction

The role of matrix metalloproteinases (MMPs) in type 2 diabetes mellitus (DM) is not clear as increased activation of MMPs in the vasculature contrasts with decreased activity of MMPs in the kidneys, contributing to development of nephropathy.

Material and methods

We measured serum MMP-2 and MMP-9 in 22 subjects with type 2 DM age (mean ± SD) 56.7 ±16.8 years, BMI 31.8 ±4.6 kg/m², HbA_1c 8.45 ±1.78% and in 32 controls, age 39.2 ±16.0 years, BMI 35.2 ±8.5 kg/m². In 15 subjects with 2 DM we also measured MMP-2 and MMP-9 at discharge from hospital and after 3 months (n = 8). In controls, MMP-2 and -9 were also measured during 75 g oral glucose tolerance test (OGTT).

Results

Concentrations of MMP-2 and MMP-9 were lower in subjects with type 2 DM (219 ±62 ng/ml vs. 305 ±63 ng/ml and 716 ±469 ng/ml vs. 1285 ±470 ng/ml, for MMP-2 and MMP-9, respectively, p < 0.05). MMP-9 concentrations fell at 120 min of OGTT from 1675 ±372 ng/ml to 1276 ±422 ng/ml (p < 0.05). In diabetic subjects there was a correlation between MMP-9 and HbA_1c (r = 0.51, p< 0.05). In subjects with diabetes there was a fall of HbA_1c from 9.77 ±1.76% to 8.36 ±1.54% (p < 0.01), at three months post-discharge. There was no difference in MMP-2, but there was a fall in MMP-9 at three months post-discharge in comparison to concentrations observed at admission (854 ±560 ng/ml vs. 500 ±235 ng/ml, p= 0.02).

Conclusions

Matrix metalloproteinases in type 2 and MMP-9 concentrations were lower in subjects with 2 DM than in non-diabetic controls. Regulation of MMPs appears to be complex as hyperglycaemia during OGTT results in a decrease in MMP-9, while chronic hyperglycaemia, reflected by HbA_1c, correlates with MMP-9 concentrations in subjects with 2 DM.