Use of a metallochromic indicator to study intracellular calcium movements in skeletal muscle

The transient increase in free myoplasmic calcium concentration due to depolarization of a skeletal muscle fibre is the net result of the release of calcium from the sarcoplasmic reticulum (SR) and its simultaneous removal by binding to various sites and by reuptake into the SR. We review here procedures recently developed in this laboratory for empirically characterizing the calcium removal processes in voltage-clamped fibres and for using such characterization to determine the time course of SR calcium release during a depolarizing pulse.     

Time course of calcium release and removal in skeletal muscle fibers.

The transient increase in free myoplasmic calcium concentration due to depolarization of a skeletal muscle fiber is the net result of the release of calcium from the sarcoplasmic reticulum (SR) and its simultaneous removal by binding to various sites and by reuptake into the SR. We present a procedure for empirically characterizing the calcium removal processes in voltage-clamped fibers and for using such characterization to determine the time course of SR calcium release during a depolarizing pulse. Our results reveal a decline of the SR calcium release rate during depolarization that was not anticipated from simple inspection of the calcium transients.     

Microinjection of strong calcium buffers suppresses the peak of calcium release during depolarization in frog skeletal muscle fibers

The effects of high intracellular concentrations of various calcium buffers on the myoplasmic calcium transient and on the rate of release of calcium (Rrel) from the sarcoplasmic reticulum (SR) were studied in voltage-clamped frog skeletal muscle fibers. The changes in intracellular calcium concentration (delta[Ca2+]) for 200-ms pulses to 0-20 mV were recorded before and after the injection of the calcium buffer and the underlying Rrel was calculated. If the buffer concentration after the injection was high, the initial rate of rise of the calcium transient was slower after injection than before and was followed by a slow increase of [Ca2+] that resembled a ramp. The increase in myoplasmic [Mg2+] that accompanies the calcium transient in control was suppressed after the injection and a slight decrease was observed instead. After the injection the buffer concentration in the voltage-clamped segment of the fiber decreased as the buffer diffused away toward the open ends. The calculated apparent diffusion coefficient for fura-2 (Dapp = 0.40 +/- 0.03 x 10(-6) cm2/s, mean +/- SEM, n = 6) suggests that approximately 65-70% of the indicator was bound to relatively immobile intracellular constituents. As the concentration of the injected buffer decreased, the above effects were reversed. The changes in delta[Ca2+] were underlined by characteristic modification of Rrel. The early peak component was suppressed or completely eliminated; thus, Rrel rose monotonically to a maintained steady level if corrected for depletion. If Rrel was expressed as percentage of SR calcium content, the steady level after injection did not differ significantly from that before. Control injections of anisidine, to the concentration that eliminated the peak of Rrel when high affinity buffers were used, had only a minor effect on Rrel, the peak was suppressed by 26 +/- 5% (mean +/- SE, n = 6), and the steady level remained unchanged. Thus, the peak component of Rrel is dependent on a rise in myoplasmic [Ca2+], consistent with calcium-induced calcium release, whereas the steady component of Rrel is independent of myoplasmic [Ca2+].     

Effects of low myoplasmic Mg2+ on calcium binding by parvalbumin and calcium uptake by the sarcoplasmic reticulum in frog skeletal muscle.

The effects of low intracellular free Mg2+ on the myoplasmic calcium removal properties of skeletal muscle were studied in voltage-clamped frog skeletal muscle fibers by analyzing the changes in intracellular calcium and magnesium due to membrane depolarization under various conditions of internal free [Mg2+]. Batches of fibers were internally equilibrated with cut end solutions containing two calcium indicators, antipyrylazo III (AP III) and fura-2, and different concentrations of free Mg2+ (25 microM-1 mM) obtained by adding appropriate total amounts of ATP and magnesium to the solutions. Changes in AP III absorbance were used to monitor [Ca2+] and [Mg2+] transients, whereas fura-2 fluorescence was mostly used to monitor resting [Ca2+]. Shortly after applying an internal solution containing less than 60 microM free Mg2+ to the cut ends of depolarized fibers most of the fibers exhibited spontaneous repetitive movements, suggesting that free internal Mg2+ might affect the activity of the sarcoplasmic reticulum (SR) calcium channels at rest. The spontaneous contractions generally subsided. In polarized fibers the maximal amplitude of the calcium transient elicited by a depolarizing pulse was about the same whatever the internal [Mg2+], but its decay after the end of the pulse slower in low [Mg2+]. In low [Mg2+] (less than 0.14 mM), the mean rate constant of decay obtained from fitting a single exponential plus a constant to the decay of the calcium transients was approximately 30% of its value in the control fibers (1 mM internal [Mg2+]). A model characterizing the main calcium removal properties of a frog skeletal muscle fiber, including the SR pump and the Ca-Mg sites on parvalbumin, was fitted to the decay of the calcium transients. Results of the fits show that in low internal [Mg2+] the slowing of the decay of the calcium transient can be well predicted by both a decreased rate of SR calcium uptake and an expected decreased resting magnesium occupancy of parvalbumin leading to a reduced contribution of parvalbumin to the overall rate of calcium removal. These results are thus consistent with the known properties of parvalbumin as a Ca-Mg buffer and furthermore suggest that in an intact portion of a muscle fiber, the activity of the SR calcium pump can be affected by the level of free Mg2+.     

A general procedure for determining the rate of calcium release from the sarcoplasmic reticulum in skeletal muscle fibers.

A general procedure for using myoplasmic calcium transients measured with a metallochromic indicator dye to calculate the time course of calcium release from the sarcoplasmic reticulum in voltage-clamped skeletal muscle fibers is described and analyzed. Explicit properties are first assigned to all relatively rapidly equilibrating calcium binding sites in the myoplasm so that the calcium content (CaF) in this pool of "fast" calcium can be calculated from the calcium transient. The overall properties of the transport systems and relatively slowly equilibrating binding sites that remove calcium from CaF are then characterized experimentally from the decay of CaF following fiber repolarization. The rate of calcium release can then be calculated as dCaF/dt plus the rate of removal of calcium from CaF. Two alternatives are assumed for the component of CaF that is due to fast binding sites intrinsic to the fiber: a linear instantaneous buffer or a set of binding sites having properties similar to thin filament troponin. Both assumptions yielded similar calcium release wave forms. Three alternative methods for characterizing the removal system are presented. The choice among these or other methods for characterizing removal can be based entirely on convenience since any method that reproduces the decay of CaF following fiber repolarization will give the same release wave form. The calculated release wave form will be accurate provided that the properties assumed for CaF are correct, that release turns off within a relatively short time after fiber repolarization, that the properties of the slow removal system are the same during and after fiber depolarization, and that possible spatial nonuniformities of free or bound calcium do not introduce major errors.     

Calcium transients and calcium binding to troponin at the contraction threshold in skeletal muscle.

Antipyrylazo III calcium transients from voltage-clamped, cut skeletal muscle fibers of the frog were recorded, and the calcium binding to the regulatory sites of troponin C was calculated. The strength-duration curve for the contraction threshold was determined. It was found that the increase in myoplasmic calcium concentration necessary to produce the same level of contractile activation, i.e., the just visible movement, was approximately 60% higher at more positive membrane potentials resulting from short depolarizing pulses than at rheobase. However, using biochemical data for the kON and kOFF rate coefficients of the binding sites, the calculated maximums of the calcium binding curves were about the same at different voltages, and the time to maximum saturation was roughly equal to the latency of the contractions. To characterize the calcium binding in intact fibers more accurately, those values of the kON and kOFF rate coefficients that gave equal peak saturations during threshold movement at different membrane potentials were determined.     

Desensitization to norepinephrine includes refractoriness of calcium release in myocardial cells

Localization of myoplasmic free calcium was measured in fura2-loaded single rat myocardial cells to determine whether the mechanism of norepinephrine desensitization includes redistribution of calcium. Fluorescence intensities at each pixel were quantitated by use of a photon-counting, microchannel plate camera. From these images, values of calcium-dependent fluorescence intensity averages in whole cells, areas of calcium release (as zones of high intracellular calcium concentrations), and ratios of fluorescence intensity in central vs. peripheral sites were determined. Stimulation by 1 nM norepinephrine caused an increase in total free intracellular calcium and an activation of intracellular calcium release sites from subsarcolemmal pools initially and later from centrally located calcium pools. Subsequent addition of 100 nM norepinephrine failed to cause significant intracellular calcium release from centrally located pools. In contrast, forskolin exposure still released high concentrations of calcium from these central pools. These results indicate that pretreatment with even a relatively small concentration of norepinephrine causes markedly decreased subsequent intracellular calcium release from centrally located sarcoplasmic reticulum because of a refractoriness of the link between receptor activation and calcium release.     

Low myoplasmic Mg2+ potentiates calcium release during depolarization of frog skeletal muscle fibers.

The role of intracellular free magnesium concentration ([Mg2+]) in modulating calcium release from the sarcoplasmic reticulum (SR) was studied in voltage-clamped frog cut skeletal muscle fibers equilibrated with cut end solutions containing two calcium indicators, fura-2 and antipyrylazo III (AP III), and various concentrations of free Mg2+ (25 microM-1 mM) obtained by adding appropriate total amounts of ATP and magnesium to the solutions. Changes in AP III absorbance were used to monitor calcium transients, whereas fura-2 fluorescence was used to monitor resting calcium. The rate of release (Rrel) of calcium from the SR was calculated from the calcium transient and found to be increased in low internal [Mg2+]. After correcting for effects of calcium depletion from the SR and normalization to SR content, the mean values of the inactivatable and noninactivatable components of Rrel were increased by 163 and 46%, respectively, in low Mg2+. Independent of normalization to SR content, the ratio of inactivatable to noninactivatable components of Rrel was increased in low internal [Mg2+]. Both observations suggest that internal [Mg2+] preferentially modulates the inactivatable component of Rrel, which is thought to be due to calcium-induced calcium release from the SR. This could also explain the observation that, in low internal [Mg2+], the time to the peak of the calcium transient for a 5-ms depolarizing pulse was not very different from the time to the peak of the delta [Ca2+] for a 10-ms pulse of the same amplitude. Finally, in low internal [Mg2+], the calcium transient elicited by a short depolarizing pulse was in some cases clearly followed by a very slow rise of calcium after the end of the pulse. The observed effects of reduced [Mg2+] on calcium release are consistent with a removal of the inhibition that the normal 1 mM myoplasmic [Mg2+] exerts on calcium release in skeletal muscle fibers.     

Growth and ion uptake by maize seedlings on solutions variable in calcium and flow rate

Summary Growth and ion uptake by maize seedlings was studied as functions of calcium concentration and rate of flow of nutrient solutions. With calcium concentrations established at 1, 10, and 100 ppm Ca, all other nutrients being kept at constant concentration, the solutions passed through narrow culture trays at velocities of 2, 8, and 24 liter/day.Fresh and dry weights of seedlings increased as the concentration of calcium increased from 1 to 10 or 100 ppm for any value ofV.Positive response in the plant calcium content was particularly noticed when Ca increased to 100 ppm in the nutrient solution. Phosphorus content of tops decreased with increasing (Ca) in the nutrient solution, while the other elements were more or less unaffected. In the case of roots, phosphorus was generally increased, iron decreased and potassium showed inconsistent trends.Estimates of the uptake efficiency relative to calcium were made by calculating the ratio of the amount of calcium taken up by the entire seedling to the product of calcium concentration and daily flow rate of the solution. It appears that the uptake efficiency diminishes with increasing flow rate. Other instances points to the maintenance of the same uptake efficiency as (Ca) increased with the decreasingV or vice versa. These observations point to the critical nature of environmental factors on the effective contacts between supply and uptake belts that have been adopted as model in this study.     

On the mechanisms of ATP-induced and succinate-induced redistribution of cations in isolated rat liver cells

1. The ability of external ATP to induce calcium uptake in isolated rat liver cells was further characterized. Stimulation of calcium uptake was specific for ATP, other nucleotides or ATP metabolites had no comparable effect. ATP was dephosphorylated while stimulating calcium uptake, but there was no stoichiometry between ATP hydrolysis and calcium uptake nor did dephosphorylation depend on calcium concentration. ATP acted from outside and was dephosphorylated by an ecto-ATPase of the cells. 2. In addition to its direct action, ATP enhanced succinate-dependent calcium uptake in a cooperative fashion. This is best explained by different sites of action. ATP increases cell membrane permeability while succinate stimulates uptake into mitochondria. 3. ATP was able to lower Na+ and K+ gradients and the pH gradient between cells and incubation medium. Increasing calcium concentration counteracted this effect though calcium uptake was then stimulated. 4. Succinate alone did not affect monovalent cation gradients but raised the pH gradient. It partially counteracted the ATP effects on these gradients. 5. Since catecholamine-like actions of ATP may be mediated by an increase in cytoplasmic calcium concentration, the action of extracellular ATP can be taken as a model to study the role of calcium as a transmitter of hormone actions. From interdependence between ATP-stimulated and succinate-stimulated calcium uptake, conclusions can be drawn on the resulting cytoplasmic calcium concentration and its effect on plasma membrane permeability.     

Dependence of ionophore- and caffeine-induced calcium release from sarcoplasmic reticulum vesicles on external and internal calcium ion concentrations.

The effects of the ionophore, X537A, and caffeine on ATP-dependent calcium transport by fragmented sarcoplasmic reticulum were studied in the absence (calcium storage) or presence (calcium uptake) of calcium-precipitating anions. The ionophore caused rapid calcium release after calcium storage, the final level of calcium storage being the same whether a given concentration of X537A was added prior to initiation of the reaction or after calcium storage had reached a steady state. Although 10 to 12 muM X537A caused approximately 90% inhibition of oxalate-supported calcium uptake when added prior to the start of the reaction, this ionophore concentration caused only a small calcium release when added after a calcium oxalate precipitate had formed within the vesicles, and only slight inhibition of calcium uptake velocity when added during the calcium uptake reaction. When low initial calcium loads limited calcium uptake to 0.4 mumol of calcium/mg of protein, subsequent calcium additions in the absence of the ionophore led to renewed calcium uptake. Uptake of the subsequent calcium additions was not significantly inhibited by 10 to 12 muM X537A. These phenomena are most readily understood in terms of constraints imposed by fixed Cai (calcium ion concentration inside the vesicles) on the pump-leak situation in sarcoplasmic reticulum vesicles containing a large amount of an insoluble calcium precipitate, where most of the calcium is within the vesicles and Cai is maintained at a relatively low level. These constraints restrict calcium loss after calcium permeability is increased because calcium release can end when the calcium pump is stimulated by the increased Cao (calcium concentration outside the vesicles) so as to compensate for the increased efflux rate. In contrast, an increased permeability in vesicles that have stored calcium in the absence of a calcium-precipitating ion causes a much larger portion of the internal calcium store to be released. Under these conditions calcium storage capacity is low so that release of stored calcium is less able to raise Cao to levels where the calcium pump can compensate for the increased efflux rate. The constraints imposed by anion-supported calcium uptake explain the finding that more calcium is released by X537A or caffeine when these agents are added at higher levels of Cao, and that more calcium leaves the vesicles in response to a given increase in calcium permeability at higher Cai. Although such calcium release is amplified by increased Cao, the amplification is attributable to the constraints described above and does not represent a "calcium-triggered calcium release."     

Polyamines Stimulate Mitochondrial Calcium Transport in Rat Brain

The effects of the polyamines spermine and spermidine on rat brain mitochondrial calcium transport were examined using a variety of techniques for measuring the kinetics of calcium uptake and the buffering capabilities of isolated mitochondria. Spermine both increased the rate of calcium accumulation and decreased the set-point to which isolated mitochondria buffer free calcium concentration. In the presence of physiological concentrations of sodium and magnesium, spermine lowered the extramitochondrial calcium level to approximately 0.3 microM, a value close to the resting intracellular calcium concentration. The effect of polyamines was concentration dependent, with a half-maximal effect of spermine observed at approximately 0.1-0.4 mM (respiratory substrate dependent), whereas spermidine was approximately 10 times less potent. Calcium transport by hippocampal mitochondria was stimulated markedly more by spermine than was calcium transport by mitochondria isolated from brainstem. The stimulatory effect of spermine was not due to an increase in the transport of respiratory substrates inside the mitochondria nor to an effect on the enzymes using these respiratory substrates. An examination of the effect of spermine on the kinetics of calcium uptake indicated that spermine increased calcium uptake maximally at low calcium concentrations. Beyond that level, the stimulatory effect of spermine decreases, and spermine can even inhibit calcium uptake. These results are in good agreement with previous reports on the effects of polyamines on calcium transport in mitochondria from peripheral tissue. They support the hypothesis that spermine increases the rate of calcium uptake by mitochondria by increasing the affinity of the uniporter for calcium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vitellogenesis in the cockroach Nauphoeta cinerea: Separation of two classes of ovarian binding sites and calcium effects on binding and uptake

Two classes of vitellogenin binding sites with K_d-values of 7.3 nM and 290 nM were observed in follicle-membrane preparations of the cockroach Nauphoeta cinerea using a membrane-binding assay at pH 8. Separation of follicle cells and basal laminae from oocyte membranes prior to binding studies showed that the fraction consisting of follicle cells and basal laminae (FC/BL) contained high-affinity binding sites for vitellogenin (K_d=16.6 nM), whereas loweraffinity binding sites (K_d=200 nM) were found in the oocyte membrane fraction. The concentration of Ca²⁺ had a distinct effect on vitellogenin binding and uptake: maximal binding to the oocyte membrane fraction was observed at 0.3 mM Ca²⁺ and to the FC/BL fraction at 10 mM, whereas uptake of vitellogenin by oocytes in vitro was highest at 4 mM Ca²⁺. The calcium ionophore A23187 decreased vitellogenin uptake. This effect of A23187 could be counteracted by the calcium chelator Quin2. A hypothetical model for the uptake of vitellogenin into follicles of Nauphoeta cinerea is suggested.     

Modulation of the intestinal Ca2+ uptake by a cheese whey protein digest

We have previously constructed a system which enables the search for factors that could modulate the intestinal calcium transporter, CaT1 (TRPV6; Takano et al., Cytotechnology, 43, 113 (2003)). This system evaluates the CaT1-mediated calcium uptake by using CHO cells stably expressing human CaT1 (CHO-hCaT1 cells). We found that a cheese whey protein digest (CWP-D) increased the calcium uptake by the CHO-hCaT1 cells. CWP-D also enhanced the calcium uptake in human intestinal Caco-2 cells. The in vivo effects of CWP-D were then measured by using rats with enteral feeding. Although enteral feeding decreased the portal calcium concentration, CWP-D partially suppressed the decrease, suggesting that CWP-D could be used for food to enhance calcium absorption.     

The use of flocculating brewer's yeast for Cr(III) and Pb(II) removal from residual wastewaters

The use of inexpensive biosorbents to sequester heavy metals from aqueous solutions, is one of the most promising technologies being developed to remove these toxic contaminants from wastewaters. Considering this challenge, the viability of Cr(III) and Pb(II) removal from aqueous solutions using a flocculating brewer's yeast residual biomass from a Portuguese brewing industry was studied. The influence of physicochemical factors such as medium pH, biomass concentration and the presence of a co-ion was characterised. Metal uptake kinetics and equilibrium were also analysed, considering different incubation temperatures. For both metals, uptake increased with medium pH, being maximal at 5.0. Optimal biomass concentration for the biosorption process was determined to be 4.5?g dry weight/l. In chromium and lead mixture solutions, competition for yeast binding sites was observed between the two metals, this competition being pH dependent. Yeast biomass showed higher selectivity and uptake capacity to lead. Chromium uptake kinetic was characterised as having a rapid initial step, followed by a slower one. Langmuir model describes well chromium uptake equilibrium. Lead uptake kinetics suggested the presence of mechanisms other than biosorption, possibly including its precipitation.     

Review of some unusual effects of calcium binding to fibrinogen

Calcium binding curves of human and bovine fibrinogen were obtained by using a calcium sensitive electrode. The two were identical and showed 2 high, 2-3 medium and more than 15 low affinity sites. Differential scanning calorimetry at neutral pH demonstrated the presence of the D and E domains of fibrinogen; however, at pH 3.5 the D-domain was split into two. The presence of the subdomains was demonstrated also by digestion by pepsin at this pH. Combination of digestion of fibrinogen and of its fragments with different enzymes and temperatures identified up to 12 subdomains in the original molecule. Clotting of fibrinogen by thrombin at pH 7.0 was investigated also by differential scanning calorimetry. In the absence of Ca2+ clotting elicited a 40% increase in the enthalpy of thermal denaturation of the D domain of fibrinogen, but the position of the peak increased only by 0.4 degrees C. However, with clotting in the presence of 10(-3) M calcium the former increased by 70-75% and the latter by 11.0 degrees C, while these parameters of the E-domain remained unchanged. Changes of bound calcium during clotting were also measured with the calcium sensitive electrode. These had to be corrected, because the drop in free calcium was partly compensated by release of some calcium that was already bound to fibrinogen. Log of the half time of calcium uptake plotted against log thrombin concentration indicated a first order process with respect to thrombin concentration, moreover, the rate determined corresponded to that of the conformation change measured by calorimetry. The calcium uptake was correlated with release of the fibrinopeptides. Release of fibrinopeptide B follows parallel to binding of calcium and that of fibrinopeptide A is about fourfold faster. Polymerization and formation of thick bundles of fibrin is connected with release of fibrinopeptide A. Clotting with Ancrod, an enzyme that releases only fibrinopeptide A, showed only minimal binding of calcium. The polymerization inhibiting tetrapeptide Gly-Pro-Arg-Pro also depressed binding of calcium. These data suggest that a calcium-binding site must be in the proximity of the site of release of fibrinopeptide B and of a polymerization site.     

Embryonic chick intestine in organ culture: hydrocortisone and vitamin D-mediated processes.

The effects of the glucocorticoid, hydrocortisone (HC), on vitamin D-mediated responses were examined in the organ-cultured, embryonic chick duodenum. In this system tissue responses to vitamin D-steroids in the culture medium include increased cAMP concentration, de novo synthesis of a specific calcium-binding protein (CaBP), enhanced uptake and transmucosal transport of calcium, and increased alkaline phosphatase activity. HC at levels ≥ 27.5 nm increased vitamin D-induced CaBP concentration: This apparently represents the first report of an interaction of HC with another steroid, vitamin D, in the regulation of the concentration of a specific protein. High levels of HC (≥27.5 μm) in the culture medium reduced duodenal calcium uptake and transmucosal transport regardless of the presence of vitamin D. However, at lower concentrations of HC (≤2.75 μm), only vitamin D-independent calcium uptake (basal calcium uptake) was reduced. Actinomycin D had no effect on HC reduction of basal calcium uptake suggesting that new protein synthesis is not involved in this action. In other experiments either HC or vitamin D stimulated phosphate and glucose uptake, and this uptake was potentiated by the presence of both steroids. HC also stimulated alanine uptake. Either HC or vitamin D increased both alkaline phosphatase activity (APA) and cAMP concentration, but together their activities were only additive. The data accumulated thus far indicate that HC directly influences calcium (and other nutrient) uptake by the duodenum and increases the concentration of the vitamin D-induced CaBP. Other vitamin D-mediated responses (APA and cAMP) were influenced by HC but there was no readily discernible relationship to nutrient uptake.     

Bench-scale and packed bed sorption of methylene blue using treated olive pomace and charcoal

A combination of olive pomace after solvent extraction and charcoal produced from the solid waste of olive oil press industry was used as an adsorbent for the removal of methylene blue (MB) dye from aqueous solutions. Batch tests showed that up to 80% of dye was removed when the dye concentration was 10 mg/ml and the sorbent concentration was 45 mg/ml. An increase in the olive pomace concentration resulted in greater dye removal from aqueous solution, and an increase in MB dye concentration at constant adsorbent concentration increased the dye loading per unit weigh of adsorbent. In the kinetic of the adsorbent process, the adsorption data followed the second-order kinetic model better than first order kinetic model. Charcoal showed higher sorption capacity (uptake) than that of olive pomace. In the fixed bed adsorption experiment, the breakthrough curves showed constant pattern behavior, typical of favorable isotherms. The breakthrough time increased with increasing bed height, decreasing flow rate and decreasing influent concentration and methylene blue dye uptake. The uptake of MB dye was significantly increased when a mixture of olive pomace and charcoal was packed in the column in a multi-layer fashion. Different models were used to describe the behavior of this packed-sorption process.     

Calcium uptake, release and ryanodine binding in melanosomes from retinal pigment epithelium

High levels of calcium have been reported in pigmented tissues of the vertebrate eye, such as retinal pigment epithelium (RPE). Melanin granules also have high calcium concentrations, suggesting that melanin granules may be a calcium reservoir. Here we characterized the uptake and release of calcium in a pure melanosomal fraction obtained from frog RPE. Melanosomes take up 45Ca by a saturable system with an apparent KM of 0.5 mM. About 40% of 45Ca accumulation was insensitive to low temperature. 45Ca uptake was not affected by verapamil, nifedipine, dantrolene, vanadate, thapsigargin or cyclopiazonic acid, but it was reduced by 50% by ruthenium red, and increased by the ionophore A23187 and nigericin. Release of 45Ca-loaded was stimulated by caffeine and inositol 1,4,5 trisphosphate (IP3). Caffeine stimulated release of calcium was blocked by either ryanodine or ruthenium red, but calcium released by IP3 was not affected by heparin. No binding of 3H-IP3 was observed. The 3H-ryanodine binding sites exhibited a KB of 1.3 nM and a Bmax of 12.1 fmol/mg protein. Thus, our results suggest that melanosomes may function as intracellular organelles that regulate calcium concentration in RPE.     

Intestine, bone, and mammary gland contributions to maternal plasma calcium increase after abrupt weaning.

The response of maternal plasma calcium concentration to the abrupt and permanent removal of the suckling pups on Day 13 of lactation was investigated. Maternal plasma calcium did not change at 6 hr or 12 hr following pup removal. At 18 hr and 24 hr after weaning, the maternal plasma calcium concentration increased in mothers consuming either 0.47% calcium or 0.02% calcium diets. At 24 hr after weaning, the plasma calcium increase in mothers consuming low dietary calcium was 55% that of mothers consuming adequate dietary calcium. The contribution of the mammary gland to the plasma calcium increase in rats consuming the low dietary calcium was investigated by removing the mammary glands. Following mammary gland removal, plasma calcium increased 50% compared with mothers that had intact mammary glands. The data suggest that intestinal absorption of calcium and bone calcium mobilization remain stimulated by the lactation process for at least 24 hr after removal of the nursing pups.