Ca2+ and Sr2+ activation: Comparison of cardiac and skeletal muscle contraction models

The mechanism of contraction in rabbit fast-twitch, and bovine and rabbit cardiac muscle was examined using functionally skinned fibers, ATPase activity of myofibrils, and cardiac or skeletal troponintropomyosin regulated actin heavy meromyosin. The Ca²⁺ and Sr²⁺ activation properties for the different measures of contraction were evaluated. (1) Tension in rabbit and bovine cardiac skinned fibers and rabbit cardiac myofibrillar ATPase were activated equally well by either Ca²⁺ or Sr²⁺. By contrast, rabbit adductor magnus (fast-twitch) skinned fibers required substantially higher [Sr²⁺] than [Ca²⁺] for activation, as did rabbit myofibrils from back muscle (fast-twitch). (2) Substantially more Sr²⁺ than Ca²⁺ was also required for activation of skeletal muscle actin heavy meromyosin ATPase, controlled by either the skeletal or cardiac troponin-tropomyosin complex, similar to the activation of fast-twitch muscle. (3) The absence of correlation between the divalent cation selectivity properties of actin heavy meromyosin ATPase controlled by cardiac troponin-tropomyosin and cardiac muscle tension or myofibrillar ATPase activation by Ca²⁺ and Sr²⁺ suggests that troponin, if primarily responsible for the activation of cardiac muscle, has very different in vivo and in vitro binding properties. (4) The close correlation between percentage of maximal Ca²⁺- and Sr²⁺-activated myofibrillar ATPase and tension in skinned fibers strongly justifies the use of myofibrillar ATPase, in contrast to a reconstituted troponin-tropomyosin actin heavy meromyosin ATPase system, as a biochemical measure of contraction.     

Quantification of des-Arg-bradykinin using a chemiluminescence enzyme immunoassay: application to its kinetic profile during plasma activation

There is a renewed interest in the kininase I pathway of kinin metabolism, because des-Arg⁹-bradykinin (des-Arg⁹-BK) and des-Arg¹⁰-Lys-BK are selective and potent agonists of the B₁ receptors, that are apparently upregulated by tissue injury. We have developed a polyclonal rabbit antiserum against des-Arg¹⁰-Lys-BK. In a radioimmunoassay for des-Arg¹⁰-Lys-BK, this antiserum exhibited high specificity. Notably, native kinins with the C-terminal Arg residue, bradykinin (BK) and Lys-BK, did not cross-react to a significant extent, whereas des-Arg⁹-BK and digoxygenin (DIG)-des-Arg⁹-BK exhibited a complete cross-reactivity. The antibodies were used to set up a sensitive chemiluminescence enzyme immunoassay (CLEIA) using the DIG-anti-DIG system as intermediate for the revelation of the immune complexes. The detection limit and the half-maximal saturation concentration for des-Arg⁹-BK were 27 and 1530 fmol/ml respectively. This assay, as well as another for BK quantification, have been applied in vitro to rabbit plasma activated by kaolin. The conversion of BK into des-Arg⁹-BK was generally efficient, and the persistence and concentration of both peptides were increased in the presence of enalaprilat an inhibitor of the angiotensin converting enzyme (ACEI). Rabbits treated with bacterial lipopolysaccharide exhibited and increase of plasma immunoreactive des-Arg⁹-BK that was potentiated in animals also treated with ACEI. This CLEIA for des-Arg⁹-BK is a new analytical tool applicable to analyze of the kininase I metabolites of kinins in vitro and in vivo. Measurements of des-Arg⁹-BK may be useful indicators of the kallikrein-kinin system activation.     

Long term spectrotypic and idiotypic stability of thyroglobulin autoantibodies in patients with Hashimoto''s thyroiditis.

Isoelectric focusing of serial bleeds from patients with Hashimoto's thyroiditis was carried out and thyroglobulin (Tg) autoantibodies visualized using 125I-labelled Tg followed by autoradiography. Although the spectrotype of these antibodies was polyclonal and varied from patient to patient, each individual's spectrotype remained constant during the disease. Similar results were obtained if immunoblots were stained with rabbit anti-idiotype (anti-Id) raised to these autoantibodies. Using radioimmunoassay (RIA), it is shown that the levels of Id remain constant over several years whether assayed on crude immunoglobulin (Ig) fractions or affinity-purified anti-Tg. Therefore, once established, the autoimmune disease appears to be stable in terms of autoantibody spectrotype and idiotype in the patients studied.     

A Highly Sensitive Radioimmunoassay for Human Growth Hormone Using a Monoclonal Antibody

Biozzi-strain mice were immunized with a highly purified preparation of 20K wriant of hGH. Spleen-cells were fused with SP2/0Ag14 myeloma cells. Clone productions were screened for specificity toward 20K and 22K hGH and for the affinity constant of antibody-antigen reaction. For the selected monoclonal antibody, Ka was 1.02.10¹¹ L/M using 22K hGH as both tracer and reference preparation. No cross reactivity was found with PRL and other pituitary hormones; hPL reactivity was 0.002 percent that of hGH. According to these antibody characteristics, a highly sensitive RIA system was developed and used for specific GH measurement in human serum. Using logit-log co-ordinates, the slope of the standard curve was -1.099 and the minimum detected dose was 0.5 uIU/ml.

Excellent correlation (r=0.9575) was found between assay data in this system and those of a conventional RIA method using specific polyclonal rabbit antiserum.

The International Reference preparation (66/217) could adequately be used to calibrate the monoclonal antibody system since the in house internal 22K GH standard and international one were equally well recognized by the monoclonal antibody.     

Molecular origin of Na/Li exchanger: Evidence against the involvement of major cloned erythrocyte transporters

Numerous studies have demonstrated heightened Na⁺/Li⁺ countertransport (NLCT) activity in erythrocytes of patients with essential hypertension or diabetic nephropathy. The same carrier also contributes to the therapeutic action of lithium salt, widely used in the treatment of psychiatric disorders. However, the molecular origin of NLCT remains unknown. This study examined the role of major ion transporters in NLCT by comparative analysis of its activity and that of ion transporters providing inwardly directed ⁸⁶Rb, ²²Na and ³²P fluxes. NLCT was below the detection limit in rat erythrocytes and ∼50-fold higher in rabbits compared to humans. Unlike NLCT, the activities of Na⁺,K⁺-ATPase, Na⁺,K⁺,2Cl⁻ cotransporter and anion exchanger were somewhat similar in the erythrocytes of these species, whereas Na⁺,P_i cotransport was in 1:2:6 proportion in rats, humans and rabbits, respectively. Loading of erythrocytes with Li⁺ for NLCT measurement did not affect the activity of Na⁺,P_i cotransporter. Keeping in mind that NLCT is much higher in rabbits vs humans and rats, we compared the set of membrane proteins in these species using 2-dimensional gel electrophoresis. This approach revealed 174 common spots, whereas 132 proteins were detected only in human and rabbit erythrocyte membranes. Among these proteins, we found 17 spots whose expression was higher by more than 5-fold in rabbit compared to human erythrocytes. Thus, our results argue against the involvement of major ion transporters in NLCT. They also show that comparative proteomics is a potent tool to identify the molecular origin of this carrier.     

Development and validation of an enzyme-linked immunosorbent assay for detection of cortisol in human saliva

Non-invasive measurement of cortisol in saliva is of prime importance as it represents a bioavailable neuroendocrine marker for stress. Therefore, in this study, we developed an enzyme immune assay that was suitable for salivary cortisol measurements. For that purpose, rabbit polyclonal antibody was raised against cortisol-3-CMO:BSA conjugate. The test was based on competition of liquid phase cortisol with conjugated cortisol on the solid phase. Primary antibody was used to bind available sites on the conjugate, which was proportional to numbers of cortisol in liquid phase. Biotinylated secondary anti-rabbit antibody was used to detect primary antibodies by addition of streptavidin peroxidase and substrate, respectively. Color formation was stopped and yellow color was read by a plate-reader spectrophotometer. Additionally, validated test was used to met all validation criteria including. Test developed was used to establish cortisol awakening response (CAR) in saliva samples collected in the morning after awakening (0, 15, 30, and 60^th min) from women (n = 4) and men (n = 4) at 8 or 4 different days, respectively. Diurnal cortisol levels were assessed (n = 8) at after awaking 60 min at morning, 12:00, 19:00, and 22:00 hr. In conclusion, an enzyme immunoassay test was successfully produced, validated and used for cortisol measurement in saliva samples.     

Influence of Mitragyna Ciliata (MYTA) on the Microsomal Activity of ATPase Na+/K+ Dependent Extract on a Rabbit Heart

Mitragyna ciliata (MYTA) (Rubiaceae) inhibits plasmodia activity. MYTA induces a cardiotonicity of the digitalic type on rat''s isolated heart. In this work we studied the effect of MYTA on microsomal Na⁺/K⁺ dependant ATPase (Na⁺, K⁺ ATPase) extracted from the heart of a rabbit since digitalics inhibit Na⁺, K⁺ ATPase. Our results revealed that the Na⁺/K⁺ ATPase has an optimum pH of 7.4 and temperature of 37°C respectively. There is a linear relationship between the organic phosphate formed and the incubation time over 25 mins incubation period. The ATP hydrolysis rate in the presence of MYTA was 0.775 µM/min. LINEWEAVER and BURK plots showed that MYTA did not alter K_M (1.31 mM) but decreased V_MAX. This study shows that MYTA exerts a non-competitive inhibition on the microsomal Na⁺/K⁺ ATPase extracted from rabbit heart with a Ci₅₀ of 48 µg / ml. We conclude that the mechanism of action of MYTA is linked to the inhibition of the Na⁺/K⁺ ATPase like cardiotonics of the digitalic type.     

Development of a high capacity radioimmunoassay procedure: ovine follicle stimulating hormone determination in blood plasma as a model.

A sensitive, specific and accurate homologous radioimmunoassay (RIA) for ovine follicle stimulating hormone (oFSH) has been developed, using a [125I]oFSH tracer and a polyclonal rabbit anti-OFSH-serum at a final dilution of 1:224,000. The separation of free and antibody-bound tracer is based on the double antibody solid phase system. The assay was found to be specific for oFSH; cross-reactivity with oLH, oPrl and oGH was lower than 0.2%. The detection limit was 7 pg per tube. Inter- and intra- assay coefficients of variation (CV) were 3.7-8.5% and 6.6%, respectively. The use of a few selfmade appliances in combination with a well-considered time-schedule enabled the processing of three thousand blood plasma samples in triplicate within two weeks. The particular advantage of the method described here is the fast, easy and safe separation of free and antibody-bound tracer with minimal handling of radioactive tubes.     

Effect of Conjugation of Cow Milk Whey Protein with Polyethylene Glycol on Changes in their Immunoreactive and Allergic Properties

Polyethylene glycol (PEG) was conjugated with milk proteins to determine the changes of the immunoreactive properties of α-lactalbumin (α-la) and β-lactoglobulin (β-lg). The competitive ELISA method was applied to determine the antigenicity of modified protein, using rabbit polyclonal antibodies. The immunoreactivity decreased to 0.90 μg ml-1 α-la (which corresponded with 0.05%) and 0.14 μg ml^-1 β-lg (which corresponded with 0.08% of the immunoreactivity observed in raw milk). The determination of allergenicity of modified whey proteins was carried out on a group of 10 allergic patients and it was insignificantly weaker than that of a control sample of milk allergens.     

Torasemide inhibits NaCl reabsorption in the thick ascending limb of the loop of Henle

Torasemide (1-isopropyl-(4-(3-methylphenylamino)pyrid-3-yl)urea) is a new diuretic. The present study examines the effects of this substance in the isolated perfused thick ascending limb (TAL) of mouse and rabbit kidney. In cortical TAL segments of the rabbit, torasemide added to the lumen perfusate led to a fall in equivalent short circuit current (= transepithelial voltage divided by transepithelial resistance, which corresponds to the rate of chloride reabsorption) with a half maximal inhibition concentration of 3 · 10^–7 mol/l. This effect was accompanied by a hyperpolarization of the luminal and basolateral membrane from –78 to –81 mV and from –72 to –81 mV, respectively. A similar hyperpolarization of both membrane voltages was also observed in medullary TAL segments of the mouse. Torasemide, added to the basolateral perfusate of cortical TAL segments of the rabbit, also inhibited the equivalent short circuit current. However, 3 · 10^–5 mol/l were necessary for a half maximal inhibition. The fall in the equivalent short circuit current was accompanied by a significant increase in transepithelial resistance from 34 to 38 cm², by an increase in the fractional resistance of the basolateral membrane, and by a hyperpolarization mainly of the basolateral membrane. Again, similar results were obtained in the medullary TAL segment of the mouse.The strong inhibitory effect of torasemide from the lumen side can be explained by an interference with the Na⁺ 2Cl^–K⁺ carrier in the luminal membrane. In fact, torasemide apparently is structurally related to furosemide. The weaker effect of torasemide from the peritubular side can, at least in part, be explained as an interference with chloride channels present in the basolateral membrane. Torasemide is also structurally related to chloride channel blockers such as diphenylamine-2-carboxylate.Supported by Deutsche Forschungsgemeinschaft DFG Gr 480/6-4 and 6-5     

D600 blocks the Ca2+ channel from the outer surface of smooth muscle cell membrane of the rabbit intestine and portal vein

The voltage dependent Ca²⁺ inward current in single smooth muscle cells dispersed from the longitudinal muscle layer of the rabbit ileum and rabbit portal vein was recorded using the whole-cell voltage clamp technique. D600 added to the bathing solution inhibited the Ca²⁺ current, while the intracellular perfusion of this agent did not reduce the amplitude of this current. Thus, D600 probably acts from the outer surface of the membrane. The nature of the Ca²⁺ channel in smooth muscle cells seems to differ from that in cardiac muscle cells.     

Evidence for contribution of Ca2+ storage sites on unitary K+ channel currents in inside-out membrane of rabbit portal vein

While making use of the inside-out membrane patch, we examined the effects of caffeine and heparin on unitary currents of the large conductance Ca²⁺ -dependent K⁺ (maxi-K⁺) channel in the rabbit portal vein. About half of the inside-out membranes we used contained a functional Ca²⁺ -store site which facilitated modification of the maxi-K⁺ channel.When high-K⁺ solution containing 0.05mM EGTA was superfused in the bath, simultaneous openings of more than 20 maxi-K⁺ channels were observed in 39 of 83 patch membranes, and multi-channel opening appeared periodically or continuously at the holding potential of – 10mV. Most channel activities of these patch membranes were inhibited by caffeine or heparin, and some heparin-insensitive channel activities were inhibited by caffeine. The remaining patch membranes (44 out of 83) showed low activity of the maxi-K⁺ channel, and neither caffeine nor heparin modified channel activity.Therefore, in our experimental set-up, half the number of excised patch membranes contained a Ca²⁺ store site. Most Ca²⁺ store sites have inositol 1,4,5-trisphosphate (InsP₃)-activated Ca²⁺ release (IACR) and caffeine-activated Ca²⁺ release (CACR) channels and few lack the IACR channel. The mechanisms of activation of the maxi-K⁺ channel in relation to release of Ca²⁺ from the store sites can be examined in detail using the approaches we have described.     

Caffeine and histamine-induced oscillations of K(Ca) current in single smooth muscle cells of rabbit cerebral artery

In the present experiment, we characterized the intracellular Ca²⁺ oscillations induced by caffeine (1 mM) or histamine (1–3 M) in voltage-clamped single smooth muscle cells of rabbit cerebral (basilar) artery. Superfusion of caffeine or histamine induced periodic oscillations of large whole-cell K⁺ current with fairly uniform amplitudes and intervals. The oscillatory K⁺ current was abolished by inclusion of ethylenebis(oxonitrilo)tetraacetate (EGTA, 5 mM) in the pipette solution. Caffeine- and histamine-induced periodic activation of the large-conductance Ca²⁺-activated K⁺ [K_(Ca)] channel was recorded in the cell-attached patch mode. These results suggest that the oscillations of K⁺ current are carried by the K_(Ca) channel and reflect the oscillations of intracellular Ca²⁺ concentration ([Ca²⁺]_i). Ryanodine (1–10 M) abolished both caffeine- and histamine-induced oscillations. Caffeine- induced oscillations were abolished by the sarcoplasmic reticulum Ca²⁺-adenosine 5-triphosphatase (Ca²⁺-ATPase) inhibitor, cyclopiazonic acid (10 M), and a high concentration of caffeine (10 mM). Inclusion of heparin (3 mg/ml) in the pipette solution blocked histamine-induced oscillations, but did not block caffeine-induced oscillations. By the removal of extracellular Ca²⁺, but not by the addition of verapamil and Cd²⁺, the caffeine-induced oscillations were abolished. Increasing Ca²⁺ influx rate increased the frequencies of caffeine-induced oscillations. Spontaneous oscillations were also observed in cells that were not superfused with agonists, and had similar characteristics to the caffeine-induced oscillations. From the above results, it is concluded, that in smooth muscle cells of the rabbit cerebral (basilar) artery, ryanodine-sensitive Ca²⁺-induced Ca²⁺ release pools play key roles in the generation of caffeine- and histamine-induced intracellular Ca²⁺ oscillations.     

Measurement of intracellular42K diffusion in frog ventricular strips

The longitudinal distribution of⁴²K was measured in strips of frog ventricular muscle placed in a partitioned perfusion chamber. A radiation detector placed directly under the chamber was moved from point to point to scan the longitudinal distribution of⁴²K. The detector was focussed on a 0.6 mm segment of the strip by means of two slits and two Geiger tubes. A beta-particle from the strip was counted only if it passed through both Geiger tubes. This arrangement improved the spatial resolution and decreased the background and the sensitivity to Compton electrons. The intracellular diffusion constant measured with this system is 3.7×10^–6 cm²/s.Supported by NIH grant HL 24667     

Development of a Solid-Phase Radioimmunoassay for the Determination of Arginine-Vasopressin in Urine

A new solid-phase radioimmunoassay (RIA) has been developed for measuring arginine-vasopressin (AVP) in urine. AVP is first extracted from urine by adsorption on Vycor glass powder and eluted with acetone-water (60:40). The mean recovery is 75.3 ± 2.2% (n =18). The organic extract is evaporated to dryness and reconstituted in the assay buffer. Aliquots of this extract are then incubated with ¹²⁵I-AVP in polystyrene LKB tubes previously coated with the antiserum (1:50000) for 48 hours. The free radioactive fraction is removed by aspiration and the tubes are counted. Values correlate well with those obtained by liquid-phase RIA using dextran-charcoal. Urinary AVP concentrations in normal Sprague-Dawley rats and rats with varying degrees of hydration have been measured.     

Detection of Fcγ receptors on human lymphoblastoid cell surfaces using a simple solid phase radioimmunoassay specific for human IgG

The aim of this work was detect Fcγ receptors on human lymphoblastoid cells using a solid phase radioimmunoassay (RIA), specific to human IgG.RIA was performed by coupling rabbit anti-human IgG (AHIgG) to acrylamide beads to make immunobeads. The specificity and sensitivity of binding of human [¹²⁵I]IgG to immunobeads permitted detection of as little as 10^?10 M unlabeled human IgG or aggregated human IgG (AHIgG) in competitive assay.The cells were incubated with an AHIgG concentration (3 × 10^?10 M) giving 25% inhibition in the RIA; unbound AHIgG concentration was then measured in the cell supernatants using RIA. Results show that Fcγ receptors could be detected at 20°C or at 37°C (but not at 4°C) on the four B cell lines tested. At whatever temperature of incubation, Fcγ receptors were not detected on three T cell lines nor on two ‘non T-non B’ cell lines. This method allows screening of a large number of Fcγ receptor positive cells. It is also useful for detecting Fcγ receptors in membrane preparations or in detergent extracts of human B cell membranes.     

Ion transport and electrophysiology of the early proximal colon of rabbit

The ion transport properties of the mammalian descending colon have been the subject of numerous investigations during the last decade. In contrast, relatively few studies have investigated proximal segments of this organ. In the present study, we assessed transepithelial transport of Na⁺, K⁺ and Cl^– in the isolated initial segment (P1) of rabbit colon in vitro using radioisotopic tracer fluxes and electrophysiological techniques. Like the rabbit descending colon, the proximal colon actively absorbs sodium and chloride, howeveer, its transport systems are markedly different. In vivo, this segment absorbs potassium, however in vitro active potassium secretion was observed. Unlike the descending colon, Na⁺ absorption is relatively insensitive to amiloride and only a slight inhibition was obtained even at 1 mM concentrations of this drug. Na⁺ and Cl^– absorption appeared to be coupled (directly or indicrectly) since the absorption of each ion was inhibited by the removal of the other. Serosal ouabain also inhibited Na⁺ and Cl^– absorption and net K⁺ secretion. Unlike the descending colon, the proximal P1 segment did not have a net absorptive K⁺ transport system that was detectable in the presence of ouabain. Electrically, the early proximal colon has a low transepithelial resistance compared to descending colon (R _T=133±7 cm²) but a larger short-circuit current (l _sc=178±12 A/cm²). The transepithelial potential averaged –21±1 mV, in excellent agreement with values measured in vivo. The apical and basolateral membrane potentials averaged –21±1 mV and –42±1 mV and intracellular potassium activity was 70±2 mM. The findings indicate active K⁺ uptake across the basolateral membrane and passive exit across the apical membrane. The basolateral membrane conductance may be a potassium conductance that is blockable by barium. It is likely that K⁺ transport normally occurs by both cellular and paracellular routes in this epithelium. Because of the numerous differences between this segment and the descending colon, we conclude that the P1 segment of proximal colon has a distinct function in colonic electrolyte transport     

Variations of membrane cholesterol alter the kinetics of Ca2+-dependent K+ channels and membrane fluidity in vascular smooth muscle cells

The patch-clamp technique and fluorescence polarization analysis were used to study the dependence of Ca²⁺-dependent K⁺ channel kinetics and membrane fluidity on cholesterol (CHS) levels in the plasma membranes of cultured smooth muscle rabbit aortic cells. Mevinolin (MEV), a potent inhibitor of endogenous CHS biosynthesis was used to deplete the CHS content. Elevation of CHS concentration in the membrane was achieved using a CHS-enriching medium. Treatment of smooth muscle cells with MEV led to a nearly twofold increase in the rotational diffusion coefficient of DPH (D) and to about a ninefold elevation of probability of the channels being open (P _o). The addition of CHS to the cells membrane resulted in a nearly twofold decrease in D and about a twofold decrease in P _o. Elementary conductance of the channels did not change under these conditions. These data suggest that variations of the CHS content in the plasma membrane of smooth muscle cells affect the kinetic properties of Ca²⁺-dependent K⁺ channels presumably due to changes in plasma membrane fluidity. Our results give a possible explanation for the reported variability of Ca²⁺-dependent K⁺ channels kinetics in different preparations.     

Measurement by Quin2 of changes of the intracellular calcium concentration in strips of the rabbit ear artery and of the guinea-pig ileum

Ca²⁺ transient and force development were investigated in smooth muscle strips of the rabbit ear artery and the longitudinal layer of the guinea-pig ileum by using the fluorescent indicator Quin2. Agonists only transiently increased the flourescence intensity despite the enhanced contraction while excess potassium resulted in a maintained light signal. In Ca²⁺ free solutions the release by an agonist of Ca²⁺ from an intracellular store can be demonstrated. These observations illustrate the usefullness of the Ca²⁺ indicator Quin2 in the study of the excitation-contraction coupling in smooth muscle under various conditions.