Influence of temperature and cyanide on electrical and mechanical activities of isoproterenol-damaged frog heart

The electrical and mechanical activities of myocardial strips from Rana pipiens treated with isoproterenol (ISO) were studied during cyanide hypoxia at different bath temperatures (12, 25 and 35 degrees C). In normal myocardium at 12 degrees C, the action potential duration (APD) was almost unchanged but the isometric force (P) was reduced to about 60% after 30 min in 3 mmol/l NaCN. At 25 degrees C, APD and P decreased to about 80 and 60%, respectively, after exposure to cyanide for 30 min. At 35 degrees C, a fast decrease of APD (to about 30%) and P (to about 10%) was seen within 30 min. In all cases, washout of cyanide interactions was possible. Large effects occurred when ISO-damaged myocardium was exposed to cyanide. During the initial 30 min of CN-treatment, APD and P were significantly reduced in the whole temperature range between 12 and 35 degrees C, when compared with controls. When the cyanide exposure times were long enough, all preparations developed contracture. Increase of the temperature and/or ISO-pretreatment shortened the time-course for resting tension increase. The effects of cyanide on APD and resting tension (RT) were strongly correlated and presumably a result of a cyanide-induced rise of the intracellular free calcium concentration (Ca2+i).     

The semicircular canals of the hagfish Myxine glutinosa

The internal radius (r) and radius of curvature (R) of the single semicircular canals of Myxine glutinosa have unusual dimensions. In mammals and fish the increase in dimension of r and R with respect to body weight is small; in fish r is larger than in mammals of equivalent weight in order to increase the sensitivity of the canals to angular rotation and R increases correspondingly (Jones & Spells, 1963). In Myxine r is larger than in fish or mammals yet R is smaller. It is argued that the large internal radius is the result of the need to increase the sensitivity of a single canal which has to signal rotation in three planes while the small radius of curvature follows from the absence of a cupula. In order to verify that the cristae of the canals do respond to rotational velocity, recordings were obtained from the nerves serving the canals during rotation in the horizontal plane. The frequency response of several afferents recorded simultaneously at sinusoidal rotations between 0.25 and 2.0 Hz was in the form of a sine wave 90 in advance of head position, as would be expected of a velocity transducer. The gain of single afferents was an order of magnitude less than those reported for other vertebrates.     

Effects of cold cardioplegia on pH, Na, and Ca in newborn rabbit hearts

Many studies suggest myocardial ischemia-reperfusion (I/R) injury results largely from cytosolic proton (H(i))-stimulated increases in cytosolic Na (Na(i)), which cause Na/Ca exchange-mediated increases in cytosolic Ca concentration ([Ca]i). Because cold, crystalloid cardioplegia (CCC) limits [H]i, we tested the hypothesis that in newborn hearts, CCC diminishes H(i), Na(i), and Ca(i) accumulation during I/R to limit injury. NMR measured intracellular pH (pH(i)), Na(i), [Ca]i, and ATP in isolated Langendorff-perfused newborn rabbit hearts. The control ischemia protocol was 30 min for baseline perfusion, 40 min for global ischemia, and 40 min for reperfusion, all at 37 degrees C. CCC protocols were the same, except that ice-cold CCC was infused for 5 min before ischemia and heart temperature was lowered to 12 degrees C during ischemia. Normal potassium CCC solution (NKCCC) was identical to the control perfusate, except for temperature; the high potassium (HKCCC) was identical to NKCCC, except that an additional 11 mmol/l KCl was substituted isosmotically for NaCl. NKCCC and HKCCC were not significantly different for any measurement. The following were different (P < 0.05). End-ischemia pH(i) was higher in the CCC than in the control group. Similarly, CCC limited increases in Na(i) during I/R. End-ischemia Na(i) values (in meq/kg dry wt) were 115 +/- 16 in the control group, 49 +/- 13 in the NKCCC group, and 37 +/- 12 in the HKCCC group. CCC also improved [Ca]i recovery during reperfusion. After 40 min of reperfusion, [Ca](i) values (in nmol/l) were 302 +/- 50 in the control group, 145 +/- 13 in the NKCCC group, and 182 +/- 19 in the HKCCC group. CCC limited ATP depletion during ischemia and improved recovery of ATP and left ventricular developed pressure and decreased creatine kinase release during reperfusion. Surprisingly, CCC did not significantly limit [Ca]i during ischemia. The latter is explained as the result of Ca release from intracellular buffers on cooling.     

Temperature dependency of force loss and Ca(2+) homeostasis in mouse EDL muscle after eccentric contractions

The goals of this study were first to determine the effect of temperature on the force loss that results from eccentric contractions in mouse extensor digitorum longus (EDL) muscles and then to evaluate a potential role for altered Ca(2+) homeostasis explaining the greater isometric force loss observed at the higher temperatures. Isolated muscles performed five eccentric or five isometric contractions at either 15, 20, 25, 30, 33.5, or 37 degrees C. Isometric force loss, caffeine-induced force, lactate dehydrogenase (LDH) release, muscle accumulation of (45)Ca(2+) from the bathing medium, sarcoplasmic reticulum (SR) Ca(2+) uptake, and resting muscle fiber free cytosolic Ca(2+) concentration ([Ca(2+)](i)) were measured. The isometric force loss after eccentric contractions increased progressively as temperature rose; at 15 degrees C, there was no significant loss of force, but at 37 degrees C, there was a 30-39% loss of force. After eccentric contractions, caffeine-induced force was not affected by temperature nor was it different from that of control muscles at any temperature. Loss of cell membrane integrity and subsequent influx of extracellular Ca(2+) as indicated by LDH release and muscle (45)Ca(2+) accumulation, respectively, were minimal over the 15-25 degrees C range, but both increased as an exponential function of temperature between 30 and 37 degrees C. SR Ca(2+) uptake showed no impairment as temperature increased, and the eccentric contraction-induced rise in resting fiber [Ca(2+)](i) was unaffected by temperature over the 15-25 degrees C range. In conclusion, the isometric force loss after eccentric contractions is temperature dependent, but the temperature dependency does not appear to be readily explainable by alterations in Ca(2+) homeostasis.     

The temperature dependence of passive potassium permeability in mammalian erythrocytes

The effect of temperature on the "passive" permeability of mammalian plasma membranes to K+, measured as the residual flux in the presence of ouabain and bumetanide, was investigated in erythrocytes of several species. Without Ca2+ in the medium, only human red cells demonstrated the "paradoxical" rise in passive flux at low temperature (i.e., below 12 degrees C) seen by other workers. In the other species no such effect was apparent; K+ influx decreased progressively with cooling down to 0 degree C. Below 18.5 degrees C the apparent energy of activation (Ea) was very low--close to that for free diffusion in water--for red cells of all species except human. Above 18.5 degrees C the Ea was much greater and was also more variable amongst the red cells of the species chosen. Neither the inhibitors used nor cell volume changes during incubation accounted for the absence of the paradoxical effect in the species studied here. A rise in permeation of K+ with cooling can, however, be produced by the addition of Ca2+ to the medium, probably by activation of the Ca2+-sensitive K+ channel. This effect would account for previous reports of a paradoxical effect in dog and rat erythrocytes.     

Temperature dependence of Ca2+-activated K+ currents in the membrane of human erythrocytes

The currents through single Ca2+-activated K+ channels were studied in excised inside-out membrane patches of human erythrocytes. The effects of temperature on single-channel conductance, on channel gating and on activation by Ca2+ were investigated in the temperature range from 0 up to 47 degrees C. The single-channel conductance shows a continuous increase with increasing temperature; an Arrhenius plot of the conductance gives the activation energy of 29.6 +/- 0.4 kJ/mol. Reducing the temperature alters channel-gating kinetics which results in a significant increase of the probability of the channel being open (Po). The calcium dependence of Po is affected by temperature in different ways; the threshold concentration for activation by Ca2+ is not changed, the Ca2+ concentration of half-maximal channel activation is reduced from 2.1 mumol/l at 20 degrees C to 0.3 mumol/l at 0 degrees C, the saturation level of the dependence is reduced for temperatures higher then about 30 degrees C. The relevance of the obtained data for the interpretation of the results known from flux experiments on cells in suspensions is discussed.     

Immunocytochemistry of cardiac polypeptide hormones (cardiodilatin/atrial natriuretic polypeptide) in brain and hearts of Myxine glutinosa (Cyclostomata)

With the use of different region-specific antisera against partial sequences of porcine cardiodilatin (CDD)-126 and the peroxidase-antiperoxidase (PAP) technique, the central nervous system as well as the systemic and the portal vein heart of the cyclostomian species Myxine glutinosa were investigated for a possible existence of cardiac polypeptides. In contrast to mammals, CDD-immunoreactions were obtained only with antisera directed against the C-terminus of CDD (CDD 99-126) which is identical to human atrial natriuretic polypeptide (alpha hANP). CDD-immunoreactive myocardiocytes were found in high densities in the atrium of the systemic heart and in the portal vein heart. In the ventricle of the systemic heart, CDD-immunoreactive cells were extremely scarce. In agreement with the immunohistochemical results, myoendocrine cells analyzed by electronmicroscopy exhibited specific granules of an average diameter of 0.21 + 0.02 micron in equivalent localizations. Furthermore, with the use of the protein A-gold (PAG) technique, CDD-immunoreactivity was ultrastructurally localized within the specific granules of atrial myocardiocytes. In the central nervous system of Myxine glutinosa, CDD-immunoreactive perikarya and/or fibers were present on all levels from the telencephalon to the spinal cord. The results of the present study are compared with those obtained in mammals and their possible functional relevance and their meaning in phylogeny are discussed as well.     

The effect of temperature on the contractile response of cardiac muscle from two frog species, Rana temporaria and Xenopus laevis, at two different calcium concentrations-II. At pH 7.0 and pH 6.6

1. Mechanical parameters were recorded from paced ventricular cardiac muscle strips of two amphibian species. Rana temporaria and xenopus laevis. 2. The strips contracted at pH 6.6 and 7.0 with either 1.6 or 3.0 mM Ca2+ while the temperature was changed from 5 to 30 degree C. 3. In the hearts of R. temporaria, but not in those of X. laevis, in increased Ca2+ concentration at low pH could compensate for the effect of lowered pH. 4. It is concluded that during high CO2 tension and/or low pH the myocardium of X. laevis was more temperature dependent, less Ca2+ dependent in the Ca2+ range examined and less sensitive to the pH and/or the CO2 tension during physiological temperature and Ca2+ concentration, than the cardiac muscle of R. temporaria.     

Defective calcium handling in cardiomyocytes isolated from hearts subjected to ischemia-reperfusion

Although ischemia-reperfusion (I/R) has been shown to affect subcellular organelles that regulate the intracellular Ca2+ concentration ([Ca2+]i), very little information regarding the Ca2+ handling ability of cardiomyocytes obtained from I/R hearts is available. To investigate changes in [Ca2+]i due to I/R, rat hearts in vitro were subjected to 10-30 min of ischemia followed by 5-30 min of reperfusion. Cardiomyocytes from these hearts were isolated and purified; [Ca2+]i was measured by employing fura-2 microfluorometry. Reperfusion for 30 min of the 20-min ischemic hearts showed attenuated cardiac performance, whereas basal [Ca2+]i as well as the KCl-induced increase in [Ca2+]i and isoproterenol (Iso)-induced increase in [Ca2+]i in cardiomyocytes remained unaltered. On the other hand, reperfusion of the 30-min ischemic hearts for different periods revealed marked changes in cardiac function, basal [Ca2+]i, and Iso-induced increase in [Ca2+]i without any alterations in the KCl-induced increase in [Ca2+]i or S(-)-BAY K 8644-induced increase in [Ca2+]i. The I/R-induced alterations in cardiac function, basal [Ca2+]i, and Iso-induced increase in [Ca2+]i in cardiomyocytes were attenuated by an antioxidant mixture containing superoxide dismutase and catalase as well as by ischemic preconditioning. The observed changes due to I/R were simulated in hearts perfused with H2O2 for 30 min. These results suggest that abnormalities in basal [Ca2+]i as well as mobilization of [Ca2+]i upon beta-adrenoceptor stimulation in cardiomyocytes are dependent on the duration of ischemic injury to the myocardium. Furthermore, Ca2+ handling defects in cardiomyocytes appear to be mediated through oxidative stress in I/R hearts.     

Electrical and mechanical effects induced by cold temperatures in the ventricle of isolated Rana ridibunda hearts

A temperature decrease changes the contractility of the amphibian heart, but the underlying mechanisms are not totally understood. The objectives of the present work were to better understand the intrinsic mechanisms supporting contractility changes induced by a rapid temperature decrease in the ventricle of Rana ridibunda, and to investigate how fast they develop. Ventricular mechanical cycles (VMCs) and monophasic action potentials (MAPs) recorded from 15 isolated hearts were measured at 15, 30, 45, 60, 90, 120 and 150 s after the application of Ringer solutions of 20, 10 and 5 degrees C. Treatment with 10 and 5 degrees C Ringer solutions decreased the heart rate, and increased the magnitude of the ventricular contraction and the duration of the contraction and relaxation periods. The electrical changes included prolongation of the MAP depolarization plateau, which also decreased in amplitude as an effect of perfusion with 5 degrees C Ringer solution. In addition, treatment with 5 degrees C Ringer solution increased the latency of contraction. The block of L-type channels totally abolished the depolarization plateau at all perfusion temperatures, but failed to inhibit ventricular contraction. In conclusion, treatment with cold temperatures changes the electrical activity of the ventricular myocardium in R. ridibunda hearts, which results in modified ventricular contractility. Data suggest that in addition to L-type Ca2+ channels, other components that support calcium elevation are present R. ridibunda ventricular cells.     

Cryoenzymic studies on an organized system: myofibrillar ATPases and shortening.

We have exploited cryoenzymology, first, to probe the product release steps of myofibrillar ATPase under relaxing conditions and, second, to define the conditions for studying the contractile process in slow motion. Cryoenzymology implies perturbation by temperature and by the antifreeze added to allow for work at subzero temperatures. Here, we studied myofibrillar shortening and ATPases by the rapid quench flow method over a wide temperature range (-15 to 30 degrees C) in two antifreezes, 40% ethylene glycol and 20% methanol. The choice of solvent and temperature was dictated by the purpose of the experiment. Ethylene glycol (40%) is suitable for investigating the kinetics of the products release steps which is difficult in water. In this cryosolvent, the myofibrillar ATPase is not activated by Ca2+ nor is there shortening, except under special conditions, i.e., Ca2+ plus strong rigor bridges [Stehle, R., Lionne, C., Travers, F., and Barman, T. (1998) J. Muscl. Res. Cell Motil. 19, 381-392]. By the use of the glycol, we show that at low Ca2+ the kinetics of the ADP release are much faster with myofibrils than with S1. On the other hand, the kinetics of the Pi release were very similar for the two materials. Therefore, we suggest that, upon Ca2+ activation, only the Pi release kinetics are accelerated. In 20% methanol, in the presence of Ca2+, myofibrils shortened at temperatures above -2 degrees C but not below. At a given temperature above -2 degrees C, both the shortening and ATPase rates were reduced by the methanol. The temperature dependences of the myofibrillar ATPases (+/-Ca2+) converged with a decrease in temperature: at 20 degrees C, Ca2+ activated 30-fold, but at -15 degrees C, only about 5-fold. We suggest that studies in methanol may open the way for an investigation of muscle contraction in slow motion and, further, to obtain thermodynamic information on the internal forces involved in the shortening process.     

Glucose induces temperature-dependent oscillations of cytoplasmic Ca2+ in single pancreatic beta-cells related to their electrical activity

Glucose induces large amplitude oscillations of the cytoplasmic Ca2+ concentration ([Ca2+]i) in pancreatic beta-cells. The effects of temperature on these oscillations were examined by monitoring [Ca2+]i continuously in single beta-cells from ob/ob-mice using dual wavelength microfluorometry. The oscillations of [Ca2+]i disappeared when the temperature was increased above 42 degrees C and were reversibly inhibited below 30 degrees C. However, cooling did not prevent a glucose response in terms of the average rise of [Ca2+]i. Since patch clamp studies of single beta-cells have indicated a random occurrence of glucose-induced action potentials at room temperature, it was important to explore how the sugar affected the electrical activity at 37 degrees C. Using the cell-attached configuration of the patch clamp technique for such analyses, the action potentials were found to occur in bursts with durations similar to the large amplitude oscillations of [Ca2+]i.     

Ca2+/Calmodulin Distinguishes Between Guanyl-5''-yl-Imidodiphosphate-and Opiate-Mediated Inhibition of Rat Striatal Adenylate Cyclase

The inhibition of adenylate cyclase from rat striatal plasma membranes by guanyl-5'-yl-imidodiphosphate [Gpp(NH)p] and morphine was compared to determine whether Gpp(NH)p-mediated inhibition accurately reflected hormone-mediated inhibition in this system. Inhibition of adenylate cyclase activity by Gpp(NH)p and morphine was examined with respect to temperature, divalent cation concentration, and the presence of Ca2+/calmodulin (Ca2+/CaM). Gpp(NH)p-mediated inhibition was dependent on the presence of Ca2+/CaM at 24 degrees C; the inhibition was independent of Ca2+/CaM at 18 degrees C; and inhibition could not be detected in the presence, or absence, of Ca2+/CaM at 30 degrees C. In contrast, naloxone-reversible, morphine-induced inhibition of adenylate cyclase was independent of both temperature and the presence of Ca2+/CaM. Mg2+ dose-response curves also reinforced the differences in the Ca2+/CaM requirement for Gpp(NH)p- and morphine-induced inhibition. Because Gpp(NH)p-mediated inhibition was independent of Ca2+/CaM at low basal activities (i.e., 18 degrees C, or below 1 mM Mg2+) and dependent on the presence of Ca2+/CaM at higher basal activities (24 degrees C, or above 1 mM Mg2+), the inhibitory effects of Gpp(NH)p were examined at 1 mM Mg2+ in the presence of 100 nM forskolin. Under these conditions, both Gpp(NH)p- and morphine-induced inhibition of adenylate cyclase were independent of Ca2+/CaM. The results demonstrate that the requirement for Ca2+/CaM to observe Gpp(NH)p-mediated inhibition depends on the basal activity of adenylate cyclase, whereas hormone-mediated inhibition is Ca2+/CaM independent under all conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stability of equine lysozyme. I. Thermal unfolding behaviour.

The thermal denaturation of Ca(2+)- and apo-forms of equine lysozyme was followed by using far and near UV circular dichroism and intrinsic fluorescence methods. The difference found between the temperature dependence of the ellipticity at 222 nm and 287 nm, which show two stages in the thermal transition, and those at 228 nm and 294 nm, which indicate only one stage over a wide range of temperatures reflects that different subdivisions of the protein molecule are characterized by a different stability, cooperativity and pathway of denaturation. The first transition, reflected in the increase of the ellipticity at 222 nm and 287 nm, coincides with the transition detected by fluorescence and occurs at 30-50 degrees C for the apo-form and at 50-60 degrees C for the Ca(2+)-form of lysozyme. It seems to correlate with the transfer of some tryptophan residues to a more hydrophobic environment and with a local rearrangement of the tertiary and secondary structures. The unfolding transition detected by the decrease of the ellipticity at all wavelengths occurs nearly in the same temperature region for the apo- and Ca(2+)-forms, i.e. 50-80 degrees C and 55-80 degrees C, respectively. The presence of a Ca(2+)-binding loop in equine lysozyme may be partly responsible for the drastic destabilization of its structure as a whole both in the presence but especially in the absence of Ca2+ in comparison with hen and human lysozymes.     

ADP evokes biphasic Ca2+ influx in fura-2-loaded human platelets. Evidence for Ca2+ entry regulated by the intracellular Ca2+ store.

Stopped-flow fluorimetric studies at 37 degrees C have shown that ADP, at optimal concentrations, can evoke Ca2+ or Mn2+ influx in fura-2-loaded human platelets without measurable delay. In contrast, the release of Ca2+ from intracellular stores is delayed in onset by about 200 ms. By working at a lower temperature, 17 degrees C, we have now shown that the rise in cytosolic calcium concentration ([Ca2+]i) evoked by ADP in the presence of external Ca2+ is biphasic. The use of Mn2+ as a tracer for bivalent-cation entry indicates that both phases of the ADP-evoked response are associated with influx. The fast phase of the ADP-evoked rise in [Ca2+]i, which occurs without measurable delay at both 17 degrees C and 37 degrees C, is consistent with Ca2+ entry mediated by receptor-operated channels in the plasma membrane. The delayed phase, indicated by Mn2+ quench, is coincident with the discharge of the intracellular Ca2+ stores. Forskolin did not inhibit the fast phases of ADP-evoked rise in [Ca2+]i or Mn2+ quench, but completely abolished ADP-evoked discharge of the intracellular stores, the delayed phase of the rise in [Ca2+]i observed in the presence of external Ca2+ and the second phase of Mn2+ quench. The timing of the delayed event appears to be modulated by [Ca2+]i: the delayed phase of Mn2+ quench coincides with discharge of the intracellular stores in the absence of added Ca2+, but with the second phase of the ADP-evoked rise in [Ca2+]i in the presence of extracellular Ca2+. Similarly, blockade of the early phase of Ca2+ entry by SK&F 96365 further delays the second phase. It is suggested that a pathway for Ca2+ entry which is regulated by the intracellular Ca2+ store exists in platelets. This pathway operates alongside, and appears to be modulated by the activity of other routes for Ca2+ entry into the cytosol.     

The cloaca of Myxine glutinosa (Cyclostomata): a scanning electron microscopical and histochemical investigation

The cloaca of Myxine glutinosa was examined by histochemical and scanning electron microscopical methods. No copulation organ could be found in Myxine and no detectable differences in the anatomy of the cloaca between male and female Myxine glutinosa. The anal gland which is the only gland in the cloacal region is situated between rectum and ductus coelomaticus. Like the lateral mucous glands in the epidermis it consists of large mucous gland cells, thread cells and undifferentiated cells. The cloacal epithelium neither develops a spatial separation by folds nor a ciliation is present in the caudal and dorsal part of the cloacal chamber. Therefore female and male myxinoides do not show any structures which would allow transportation of sperm into the abdominal cavity or out of it.     

The effect of temperature and Ca2+ on the interaction of high-density lipoproteins with epithelial cells in the human small intestine

The effect of temperature and Ca2+ ions on the interaction of high-density lipoprotein (HDL3) with human enterocytes was studied. It was shown that Kd measured at 4 degrees C is similar to that at 37 degrees C. Maximal number of binding sites at 37 degrees C is 15-fold times higher than that at 4 degrees C. EDTA (10 mM) and CaCl2 (0.5-5 mM) did not affect binding and uptake of HDL3 by human enterocytes. The obtained results indicate that HDL3-binding with these cells depends on temperature and does not depend on Ca2+ ions.     

Multiple fatty acid sensing mechanisms operate in enteroendocrine cells: novel evidence for direct mobilization of stored calcium by cytosolic fatty acid

Fatty acids (FA) with at least 12 carbon atoms increase intracellular Ca(2+) ([Ca(2+)](i)) to stimulate cholecystokinin release from enteroendocrine cells. Using the murine enteroendocrine cell line STC-1, we investigated whether candidate intracellular pathways transduce the FA signal, or whether FA themselves act within the cell to release Ca(2+) directly from the intracellular store. STC-1 cells loaded with fura-2 were briefly (3 min) exposed to saturated FA above and below the threshold length (C(8), C(10), and C(12)). C(12), but not C(8) or C(10), induced a dose-dependent increase in [Ca(2+)](i), in the presence or absence of extracellular Ca(2+). Various signaling inhibitors, including d-myo-inositol 1,4,5-triphosphate receptor antagonists, all failed to block FA-induced Ca(2+) responses. To identify direct effects of cytosolic FA on the intracellular Ca(2+) store, [Ca(2+)](i) was measured in STC-1 cells loaded with the lower affinity Ca(2+) dye magfura-2, permeabilized by streptolysin O. In permeabilized cells, again C(12) but not C(8) or C(10), induced release of stored Ca(2+). Although C(12) released Ca(2+) in other permeabilized cell lines, only intact STC-1 cells responded to C(12) in the presence of extracellular Ca(2+). In addition, 30 min exposure to C(12) induced a sustained elevation of [Ca(2+)](i) in the presence of extracellular Ca(2+), but only a transient response in the absence of extracellular Ca(2+). These results suggest that at least two FA sensing mechanisms operate in enteroendocrine cells: intracellularly, FA (>/=C(12)) transiently induce Ca(2+) release from intracellular Ca(2+) stores. However, they also induce sustained Ca(2+) entry from the extracellular medium to maintain an elevated [Ca(2+)](i).     

Effects of cation binding on the thermal transitions in calmodulin

The thermal transitions in different forms of bovine brain calmodulin (0, 1, 2, 3 and 4 bound Ca2+ ions per molecule) have been studied by means of microcalorimetry, intrinsic tyrosine fluorescence, circular dichroism and infrared spectroscopy. The heating of the apoprotein from 5 to 110 degrees C induces at least three unfolding transitions. The heating of Ca2+-loaded calmodulin causes at least two structural transitions, one of which occurs at relatively low temperatures, from approx. 30 to approx 50 degrees C. The binding of the biologically significant Ca2+, Mg2+, Na+ and K+ ions has been measured at 12, 20, 28, 37 and 50 degrees C by means of the fluorescence method. The values of the binding parameters for these cations do not depend on temperature within the range 12 to 50 degrees C. It has been proposed that the temperature independence of the metal-ion-binding properties of calmodulin is achieved due to the temperature-induced structural changes, which adjust the protein conformation in such a way that the protein-binding parameters remain constant.     

Acute temperature change modulates the response of ICa to adrenergic stimulation in fish cardiomyocytes

The purpose of this study was to investigate how the endogenous catecholamine adrenaline protects sarcolemmal Ca(2+) flux through the L-type Ca(2+) channel (I(Ca)) during acute exposure to cold in the fish heart. We examined the response of I(Ca) to adrenergic stimulation at three temperatures (7 degrees, 14 degrees, and 21 degrees C) in atrial myocytes isolated from rainbow trout acclimated to 14 degrees C. We found that I(Ca) amplitude varied directly with test temperature and was increased by adrenergic stimulation (AD; 5 nM and 1 microM) at all temperatures. However, I(Ca) was significantly more sensitive to adrenergic stimulation at the coldest test temperature. In fact, at 7 degrees C in the absence of AD, I(Ca) was extremely low. The addition of 1 microM AD increased peak I(Ca) 7.2-fold at 7 degrees C, 2.6-fold at 14 degrees C, and 1.6-fold at 21 degrees C and ameliorated the temperature-dependent difference in Ca(2+) influx across the cell membrane. We suggest that this increased adrenergic sensitivity is a critical compensatory mechanism that allows the rainbow trout heart to maintain contractility during acute exposure to cold temperatures. In particular, the tonic level of adrenergic stimulation provided by circulating plasma catecholamines (i.e., in the nM concentration range) may be crucial for effective excitation-contraction coupling in the cold cardiomyocyte.