Evidence for two modes of Ca2+ entry following muscarinic stimulation of a human salivary epithelial cell line

Summary We have investigated muscarinic receptor-operated Ca²⁺ mobilization in a salivary epithelial cell line, HSG-PA, using an experimental approach which allows independent evaluation of intracellular Ca²⁺ release and extracellular Ca²⁺ entry. The carbachol (Cch) dose response of intracellular Ca²⁺ release indicates the involvement of a single, relatively low-affinity, muscarinic receptor site (K _0.510 or 30 m, depending on the method for [Ca²⁺] _i determination). However, similar data for Ca²⁺ entry indicate the involvement of two Cch sites, one consistent with that associated with Ca²⁺ release and a second higher affinity site withK _0.52.5 m. In addition, the Ca²⁺ entry response observed at lower concentrations of Cch (2.5 m) was completely inhibited by membrane depolarization induced with high K⁺ (>55mm) or gramicidin D (1 m), while membrane depolarization had little or no effect on Ca²⁺ entry induced by 100 m Cch. Another muscarinic agonist, oxotremorine-M (100 m; Oxo-M), like Cch, also induced an increase in the [Ca²⁺] _i of HSG-PA cells (from 72±2 to 104±5nm). This response was profoundly blocked (75%) by the inorganic Ca²⁺ channel blocker La³⁺ (25–50 m) suggesting that Oxo-M primarily mobilizes Ca²⁺ in these cells by increasing Ca²⁺ entry. Organic Ca²⁺ channel blockers (verapamil or diltiazem at 10 m, nifedipine at 1 m), had no effect on this response. The Oxo-M induced Ca²⁺ mobilization response, like that observed at lower doses of Cch, was markedly inhibited (70–90%) by membrane depolarization (high K⁺ or gramicidin D). At 100 m Cch the formation of inositol trisphosphate (IP₃) was increased 55% above basal levels. A low concentration of carbachol (1 m) elicited a smaller change in IP₃ formation (25%), similar to that seen with 100 m Oxo-M (20%). Taken together, these results suggest that there are two modes of muscarinic receptor-induced Ca²⁺ entry in HSG-PA cells. One is associated with IP₃ formation and intracellular Ca²⁺ release and is independent of membrane potential; the other is less dependent on IP₃ formation and intracellular Ca²⁺ release and is modulated by membrane potential. This latter pathway may exhibit voltage-dependent gating.     

Parameters not influenced by vesamicol: Membrane potential,calcium uptake,and internal calcium concentration of synaptosomes

In our previous study vesamicol, an inhibitor of the acetylcholine transporter of the cholinergic vesicles, inhibited veratridine-evoked external Ca²⁺-dependent acetylcholine release from striatal slices but did not influence acetylcholine release observed in Ca²⁺-free medium (4). Here we examined if the effect of veratridine on membrane potential, Ca²⁺ uptake, and intracellular Ca²⁺ concentration of synaptosomes was altered by vesamicol in parallel with the inhibition of acetylcholine release. The depolarizing effect of 10 M veratridine (from 67±2.3 mV resting membrane potential to 50.7±2.5 mV) was not significantly influenced by vesamicol (1–20 M). Vesamicol (1–20 M) had no effect on either the overall curve of the veratridine-evoked⁴⁵Ca²⁺ uptake or the amount of Ca²⁺ taken up by synaptosomes. Veratridine caused a rise in intrasynaptosomal Ca²⁺ concentration as measured by Fura2 fluorescence, and the same increase both in characteristics and in magnitude was observed in the presence of vesamicol (20 M). The K⁺-evoked (40 mM) increase of Ca²⁺ uptake and of intracellular calcium concentration were also unaltered by vesamicol. In high concentration (50 M) vesamicol inhibited both the fall in membrane potential and the elevated Ca²⁺ uptake by veratridine, indicating a possible nonspecific effect on potential-dependent Na⁺ channels at this concentration. Vesamicol, in lower concentration (20 M) when neither of the above parameters was changed, completely prevented veratridine-evoked increase of [¹⁴C]acetylcholine release. This was observed only when vesamicol was present in the media throughout the experiment after loading the preparation with [¹⁴C]choline. The results suggest that vesamicol does not interfere with veratridine-induced changes in isolated nerve terminals other than with the release of acetylcholine, thus further supporting the involvement of a vesamicol-sensitive vesicular transmitter pool in Ca²⁺-dependent veratridine-elicited acetylcholine release.     

Characterization of two different Ca2+ uptake and IP3-sensitive Ca2+ release mechanisms in microsomal Ca2+ pools of rat pancreatic acinar cells

We have examined the effect of the Ca²⁺ (Mg²⁺)-ATPase inhibitors thapsigargin (TG) and vanadate on ATP-dependent ⁴⁵Ca²⁺ uptake into IP₃-sensitive Ca²⁺ pools in isolated microsomes from rat pancreatic acinar cells. The inhibitory effect of TG was biphasic. About 40–50% of total Ca²⁺ uptake was inhibited by TG up to 10 nm (apparent K_i4.2 nm, Ca²⁺ pool I). An additional increase of inhibition up to 85–90% of total Ca²⁺ uptake could be achieved at 15 to 20 nm of TG (apparent K_i12.1 nm, Ca²⁺ pool II). The rest was due to TG-insensitive contaminating plasma membranes and could be inhibited by vanadate (apparent K_i10 m). In the absence of TG, increasing concentrations of vanadate also showed two phases of inhibition of microsomal Ca²⁺ uptake. About 30–40% of total Ca²⁺ uptake was inhibited by 100 m of vanadate (apparent K_i18 m, Ca²⁺ pool II). The remaining 60–70% could be inhibited either by vanadate at concentrations up to 1 mm (apparent K_i300 m) or by TG up to 10 nm (Ca²⁺ pool I). The amount of IP₃-induced Ca²⁺ release was constant at 25% over a wide range of Ca²⁺ filling. About 10–20% remained unreleasable by IP₃. Reduction of IP₃ releasable Ca²⁺ in the presence of inhibitors showed similar dose-response curves as Ca²⁺ uptake (apparent K_i 3.0 nm for IP₃-induced Ca²⁺ release as compared to 4.2 nm for Ca²⁺ uptake at TG up to 10 nm) indicating that the highly TG-sensitive Ca²⁺ pump fills the IP₃-sensitive Ca²⁺ pool I. At TG concentrations >10 nm which blocked Ca²⁺ pool II the apparent K_i values were 11.3 and 12.1 nm, respectively. For inhibition by vanadate up to 100 m the apparent K_i values were 18 m for Ca²⁺ uptake and 7 m for Ca²⁺ release (Ca²⁺ pool II). At vanadate concentrations up to 1 mm the apparent K_i values were 300 and 200 m, respectively (Ca²⁺ pool I). Both Ca²⁺ pools I and II also showed different sensitivities to IP₃. Dose-response curves for IP₃ in the absence of inhibitors (control) showed an apparent K_m value for IP₃ at 0.6 m. In the presence of TG (inhibition of Ca²⁺ pool I) the curve was shifted to the left with an apparent K_m for IP₃ at 0.08 m. In the presence of vanadate (inhibition of Ca²⁺ pool II), the apparent K_m for IP₃ was 2.1 m. These data allow the conclusion that there are at least three different Ca²⁺ uptake mechanisms present in pancreatic acinar cells: TG- and IP₃ insensitive but highly vanadate-sensitive Ca²⁺ uptake occurs into membrane vesicles derived from plasma membranes. Two Ca²⁺ pools with different TG-, vanadate- and IP₃-sensitivities are most likely located in the endoplasmic reticulum at different cell sites, which could have functional implications for hormonal stimulation of pancreatic acinar cells.This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 246. The authors wish to thank Dr. KlausDieter Preuß for valuable discussions and Mrs. Gabriele Mörschbächer for excellent secretarial help.     

Neuronal Control of Exocytosis and Calcium Homeostasis

We showed that 5 M acetylcholine (ACh) and 100 M norepinephrine (NE) cause increases in the total Ca²⁺ content in acinar cells by 30 and 87% and in the exocytosis intensity by 15 and 20%, respectively. Application of 5 M ACh and 100 M NE increased the free cytosolic Ca²⁺ concentration ([Ca²⁺] _i ) by 87 ± 2 and 140 ± 7 nM, respectively. Application of ACh and NE in a Ca²⁺-free external solution caused a [Ca²⁺] _i increase that was 40 and 67% lower than in physiological solution. We postulate that the exocytosis developing upon neural stimulation of the gland results from generation of Ca²⁺ transients that are spreading from the basal to the apical region of the exocrine cell, where secretory granules are concentrated.     

Effects of polymyxin B on the sarcoplasmic reticulum membrane

Polymyxin B, a cyclic peptide antibiotic, inhibits Ca²⁺-ATPase, p-nitrophenyl phosphatase and phosphorylase kinase activities associated with rabbit skeletal muscle sarcoplasmic reticulum membranes; 50% inhibition is induced by 100 M, 130M and 550 M of polymyxin respectively. The fluorescence intensity of fluorescein isothiocyanate-labeled Ca²⁺-ATPase, decreases in the presence of polymyxin (50% of the total decrease at 70 M polymyxin). On the other hand, the polypeptide inhibits calmodulin-dependent endogenous phosphorylation of 60 kDa, 20 kDa and 14 kDa membrane proteins, while an increase of calmodulin-dependent phosphorylation is observed in 132 kDa and 86 kDa proteins.     

Freeze-fracture studies on the cockroach hemocyte membrane complex: Symmetric and asymmetric membrane particle distributions

Summary Freeze-fracture studies were conducted on the membranes of normal cockroach hemocytes. The plasmalemma is asymmetric with the A fracture face containing 80–100 Å membrane intercalated particles at a concentration of 2500/². The B fracture face contains 120–150 Å particles with a relatively low density (800/²). The nuclear envelope displays an asymmetry with the A fracture face containing 1500 particles/² and the B face containing 300/ ². No significant particle size differences were observed in nuclear envelope fracture faces. Two types of symmetric membranes were also found in these cells. Both A and B fracture faces of the membrane surrounding the numerous cytoplasmic inclusion bodies contain particle sizes and concentrations similar to the B face of the plasmalemma. A second type of symmetry was observed in cells apparently engaged in exocytosis. Vesicles (0.1 D) from this process were completely particle free on both fracture faces. Such particle free vesicles could be found in the cytoplasm, attached to the plasmalemma, or completely separated from the cell.Supported by a Pharmaceutical Manufacturers Association Foundation Fellowship.The author wishes to thank Ms. Annalena K. Charla for assistance in plate preparation, Dr. Julius Schultz and the Papanicolaou Cancer Research Institute for use of the freeze-etch device, and Dr. David Smith for the electron microscope facilities.     

Somatic embryogenesis in callus cultures of Rosa hybrida L. cv. Landora

Callus was initiated from immature leaf and stem segments of rose (Rosa hybrida cv. Landora) and subcultured every four weeks on a basal medium of half-strength Murashige & Skoog (1962) salts plus 30 g l^-1 sucrose (1/2 MS) and supplemented with 2.2 M BA, 5.4 M NAA and 2.2–9.0 M 2,4-D. Embryogenic callus and subsequently somatic embryos were obtained from 8-week-old callus culture on 1/2 MS+2.2 M BA+0.05 M NAA+0.3 M GA₃+200–800 mg l^-1 L-proline. Long-term cultures were established and maintained for up to 16 months by repeated subculture of embryogenic callus on L-proline deficient medium. About 12% of cotyledonary stage embryos taken from cultures cold-stored at 8±1°C for 4 days germinated on 1/2 MS+2.2 M BA+0.3 M GA₃+24.7 M adenine sulphate.Abbreviations BA benzyladenine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid     

Alpha2-adrenoreceptor stimulation does not inhibit L-type calcium channels in mouse pancreatic β-cells

The effects of ₂-adrenergic stimulation on the Ca²⁺-current in mouse pancreatic -cells were investigated using the patch-clamp technique. When using the conventional whole-cell recording configuration (dialysis of cell interior with pipette solution), addition of adrenaline (1 M) or the ₂-adrenergic agonist clonidine (5 M) failed to reduce the Ca²⁺-current, irrespective of whether intracellular GTP (or GTP S) was present or not and at both physiological (1.3 mM) and elevated (10.2 mM) Ca²⁺-concentrations. In fact, in the absence of added guanine nucleotides, the agonists tended toincrease the Ca²⁺-current amplitude in the presence of the higher Ca²⁺-concentration. Ca²⁺-channel activation measured at 1.3 mM Ca²⁺ was not affected by clonidine. Half-maximal activation was observed at –20 mV. In addition, when Ca²⁺-currents were recorded from intact -cells, using the perforated patch whole-cell configuration, clonidine (1 M) also failed to detectably affect the Ca²⁺-current. It is therefore suggested that the inhibition of -cell electrical activity and insulin-secretion produced by ₂-adrenoreceptor stimulation does not result from suppression of the L-type Ca²⁺-current.     

The effects of triethyl lead on the development of hippocampal neurons in culture

Triethyl lead is the major metabolite of tetraethyl lead, which is used in industrial processes and as an antiknock additive to gasoline. We tested the hypothesis that low levels of triethyl lead (0.1 nmol/L to 5mol/L) interfere with the normal development of cultured E18 rat hippocampal neurons, possibly through increases in intracellular free calcium ion concentration, [Ca²⁺]_in. The study assessed survival and differentiation using morphometric analysis of individual neurons. We also looked at short-term (up to 3.75-h) changes in intracellular calcium using the calcium-sensitive dye fura-2. Survival of neurons was significantly reduced at 5 mol/L, and overall production of neurites was reduced at 2 mol/L. The length of axons and the number of axons and dendrites were reduced at 1 mol/L. Neurite branching was inhibited at 10 nmol/L for dendrites and 100 nmol/L for axons. Increases in intracellular calcium were observed during a 3.75-h exposure of newly plated neurons to 5 mol/L triethyl lead. These increases were prevented by BAPTA-AM; which clamps [Ca²⁺]_in at about 100 nmol/L. Culturing neurons with BAPTA-AM and 5 mol/L triethyl lead did not reverse the effects of triethyl lead, suggesting that elevation of [Ca²⁺]_in is not responsible for decreases in survival and neurite production. Triethyl lead has been shown to disrupt cytoskeletal elements, particularly neurofilaments, at very low levels, suggesting a possible mechanism for its inhibition of neurite branching at nanomolar concentrations.Abbreviations BAPTA-AM 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester - [Ca²⁺]_in intracellular free calcium ion concentration - DMSO dimethyl sulfoxide - E18 embryonic day 18 - FBS fetal bovine serum - fura-2AM fura-2 acetoxymethyl ester - HBSS Hanks' Balanced Salt Solution - MEM Eagle's Minimum Essential Medium     

Effect of culture medium ions on chromate reduction by resting cells of Agrobacterium radiobacter

The influence of some ions in pre-growth culture medium on chromate reduction by resting cells of Agrobacterium radiobacter strain EPS-916 was investigated. The reduction was dependent on the Fe²⁺ content of the culture medium: the higher the iron content, the lower the reduction rate. The cells showed maximum chromate reduction when pre-grown in the presence of 0.243 m Mg²⁺, 20 m Ca²⁺ and 3.6 m Mn²⁺. Chromate reduction was not affected by the addition of MgCl₂, CdCl₂, ZnCl₂, MnCl₂, Na₂SO₄ (1000 m), and Na₂MoO₄ (100 m) to the activity assays. However, activity was inhibited by the presence of Na₂SO₄ (10 mm), Na₂MoO₄ (200 m) and ferric citrate.     

Acetylation and phosphorylation of choline following high or low affinity uptake by rat cortical synaptosomes

Synaptosomal acetylcholine synthesis was found to be dependent on the presence of Na⁺-dependent HC-3 sensitive choline transport at low (5.5 mM) and high (35 mM) K⁺ concentrations. However, at 5, 20, and 100 M choline, choline phosphorylation was proportional to total choline uptake, in the presence or absence of high affinity transport. Only in the presence of eserine (50 M) did acetylcholine synthesis increase as the choline concentration was elevated from 20 M to 100 M, and this effect was observed at low and high K⁺ concentrations. Our results suggest that: 1) the synthesis of non-surplus synaptosomal ACh is dependent on high affinity choline transport; and 2) choline is equally likely to be phosphorylated after being taken up by low or high affinity transport.     

Regulatory effect of regucalcin on (Ca2+−Mg2+)-ATPase in rat liver plasma membranes: comparison with the activation by Mn2+ and Co2+

The effect of various metals and regucalcin, a calcium-binding protein isolated from rat liver cytosol, on (Ca²⁺–Mg²⁺)-ATPase activity in the plasma membranes of rat liver was investigated. Of various metals (Zn²⁺, Cu²⁺, Ni²⁺, Mn²⁺, Co²⁺ and Al³⁺; 100 M as a final concentration), Mn²⁺ and Co²⁺ increased markedly (Ca²⁺–Mg²⁺)-ATPase activity, while other metals had no effect. When Ca²⁺ was not added into enzyme reaction mixture, Mn²⁺ and Co²⁺ (25–100 M) did not significantly increase the enzyme activity, indicating that heavy metals act on Ca²⁺-stimulated phosphorylation of the enzyme. Meanwhile, regucalcin (0.25–1.0 M) caused a remarkable elevation of (Ca²⁺–Mg²⁺)-ATPase activity. This increase was not inhibited by the presence of 100 M vanadate, although the effects of Mn²⁺ and Co²⁺ (100 M) were inhibited by vanadate. Also, the inhibition of the Mn²⁺ and Co²⁺ effects by vanadate was not seen in the presence of regucalcin. Moreover, regucalcin (0.5 M) increased significantly the enzyme activity in the absence of Ca²⁺. This effect of regulcalcin was not altered by increasing concentrations of Ca²⁺ added, indicating that the regucalcin effect does not depend on Ca²⁺. The present results suggest that regucalcin activates directly (Ca²⁺–Mg²⁺)-ATPase in liver plasma membranes, and that the activation is not involved in the Ca²⁺-dependent phosphorylation of the enzyme.     

Characterization of regucalcin effect on proteolytic activity in rat liver cytosol: relation to cysteinyl-proteases

The increasing effect of regucalcin, isolated from rat liver cytosol, on neutral proteolytic activity in the hepatic cytosol was characterized. The proteolytic activity was markedly elevated by the addition of regucalcin (0.1–0.5 M) in the absence of Ca²⁺. This increase was not significantly altered by the presence of diisopropylfluorophsophate (DPF;2.5 mM)—although DFP caused a significant decrease in the proteolytic activity. Regucalcin (0.25 M) additively enhanced the dithiothreitol (DTT; 1.0 mM)—increased proteolytic activity, while the regucalcin or DTT effect was completely abolished by NEM (5 mM), indicating that regucalcin may act on the SH group in proteases. Also, regucalcin (0.25 M) enhanced the effect of Ca²⁺ (10 M) increasing liver proteolytic activity, suggesting that regucalcin does not influence on the active sites for Ca²⁺ in proteases. Moreover, the proteolytic activity of regucalcin (0.25 M) was significantly decreased by the presence of calpastatin (24 g/ml), an inhibitor of Ca²⁺-activated neutral protease (calpain). Now, regucalcin (0.25 M) increased about 7-fold the activity ofm-calpain isolated from rabbit skeletal muscle. These observations demonstrate that regucalcin directly activates cysteinyl-proteases. Regucalcin may have a role as a potent proteolytic activator in the cytoplasm of liver cells.     

Stereospecificity and electrogenicity of amino acid transport in Riccia fluitans

In the aquatic liverwort Riccia fluitans, membrane depolarization (_m), change in membrane conductance (g_m), and current-voltage (I-V) characteristics in the presence of different amino acids as well as the uptake of ¹⁴C-labeled amino acids were measured. L-isomers of the tested amino acids generate larger electrical effects (_m, g_m) than D-isomers, and the I-V characteristics show that the positive electrical inward-current of 20 mA m^-2 generated by 0.5 mM D-serine is only about 50% of the current generated by adding 0.5 mM L-serine. Whereas - and -amino acids rapidly depolarize the membrane to the same extend, with -aminobutyric acid (-AB) and dipeptides no significant electrical effects have been measured. The uptake kinetics of ¹⁴C-labeled amino acids display three components: (I) A saturable high-affinity component with K_s-values of 48 M D-alanine, 12 M -aminoisobutyric acid (AIB), 9 M L-alanine, 8 M L-proline, and 6 M L-serine, respectively; (2) an apparently linear low-affinity component, and (3) an also linear but unspecific component at concentrations >20 times the given K_s-value. Uptake of ¹⁴C-labeled AIB can be inhibited competitively by all tested neutral amino acids, the L-isomers being more effective than the D-isomers, as well as by ammonium or methylamine. Vice versa, AIB competitively inhibits uptake of L-serine and L-alanine. It is concluded that an uncharged stereospecific carrier for the investigated amino acids exists in the plasmalemma of Riccia fluitans. Accumulation ratios of about 50 suggest secondary active transport driven by a transmembrane electro-chemical gradient (mainly _m) which is generated by the electrogenic proton pump. It is suggested that this carrier binds to the amino group forming either a charged binary complex with positively charged amines (Felle 1980), or an uncharged complex with -AB or dipeptides, whereas electrogenic transport of - and -amino acids is mediated by a ternary carrier complex, probably charged by a proton.Symbols and Abbreviations _m membrane potential (mV) - E_co equilibrium potential (mV) of the transport system - g_m membrane (slope) conductance (Sm^-2) - g_m change in g_m - I-V curve current-voltage curve - AIB -aminoisobutytric acid - -AB -aminobutyric acid     

Involvement of Ca2+-stimulated adenosine 5′-triphosphatase in the Ca2+ releasing mechanism of rat liver nuclei

The regulatory role of Ca²⁺-stimulated adenosine 5-triphosphatase (Ca²⁺-ATPase) in Ca²⁺ transport system of rat liver nuclei was investigated. Ca²⁺ uptake and release were determined with a Ca²⁺ electrode. Ca²⁺-ATPase activity was calculated by subtracting Mg²⁺-ATPase activity from (Ca²⁺–Mg²⁺)-ATPase activity. The release of Ca²⁺ from the Ca²⁺-loaded nuclei was evoked progressively after Ca²⁺ uptake with 1.0 mM ATP addition, while it was only slightly in the case of 2.0 mM ATP addition, indicating that the consumption of ATP causes a leak of Ca²⁺ from the Ca²⁺-loaded nuclei. The presence of N-ethylmaleimide (NEM; 0.1 mM) caused an inhibition of nuclear Ca²⁺ uptake and induced a promotion of Ca²⁺ release from the Ca²⁺-loaded nuclei. NEM (0.1 and 0.2 mM) markedly inhibited nuclear Ca²⁺-ATPase activity. This inhibition was completely blocked by the presence of dithiothreitol (DTT; 0.1 and 0.5 mM). Also, DTT inhibited the effect of NEM (0.1 mM) on nuclear Ca²⁺ uptake and release. Meanwhile, verapamil and diltiazem (10 M), a blocker of Ca²⁺ channels, did not prevent the NAD⁺ (1.0 and 2.0 mM), zinc sulfate (1.0 and 2.5 M) and arachidonic acid (10 M)-induced increase in nuclear Ca²⁺ release, suggesting that Ca²⁺ channels do not involve on Ca²⁺ release from the nuclei. These results indicates that an inhibition of nuclear Ca²⁺-ATPase activity causes the decrease in nuclear Ca²⁺ uptake and the release of Ca²⁺ from the Ca²⁺-loaded nuclei. The present finding suggests that Ca²⁺-ATPase plays a critical role in the regulatory mechanism of Ca²⁺ uptake and release in rat liver nuclei.     

Depressed sliding velocity of isolated cardiac myosin from cardiomyopathic hamsters: Evidence for an alteration in mechanical interaction of actomyosin

We measured the relative sliding velocity of cardiomyopathic hamster cardiac myosin on actin cables by using anin vitro motility assay system. We also investigated the relationship between the velocity and both myosin isozyme content and ATPase activity. Cardiac myosin was obtained from cardiomyopathic hamsters (BIO 14.6;B) aged 3,6,9, and 18 months and age-matched controls (F1B;F). Long well-organized actin cables of an alga,Nitellopsis, wer used for the motility assay. Small latex beads (2 m in diameter) were coated with purified cardiac myosin. When myosin-coated beads were introduced into an algal cell in the presence of Mg-ATP, myosin interacted with actin and dragged the beads. Active movement of the beads along the actin cables was observed under a photomicroscope and the velocity was measured. The velocity was significantly lower in B than in F for each age group (0.47 vs. 0.71 m/s at the age of 3 months, p<0.05; 0.44 vs. 0.88 m/s at 6 months, p<0.01; 0.44 vs. 0.67 m/s at 9 months, p<0.01; 0.35 vs. 0.52 m/s at 18 months, p<0.05). Both Ca²⁺-activated ATPase activity and the percentage of -myosin heavy chain were also lower in B than in F for each age group. When examined for individual specimens, there was a positive correlation between the velocity and both myosin Ca²⁺-activated ATPase activity (r=0.84) and percentage of -myosin heavy chain (r=0.83). These data points of both control and cardiomyopathic hamsters were distributed near the regression line obtained from control and thyroxine-treated rabbits reported previously. The present results indicate that the difference in mechanical properties between control and cardiomyopathic cardiac myosin is attributed to isozyme redistribution and not to a qualitative change in each myosin molecule.     

Mechanisms of low Na+-induced increase in intracellular calcium in KCl-depolarized rat cardiomyocytes

Although low Na⁺ is known to increase the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in cardiac muscle, the exact mechanisms of low Na⁺-induced increases in [Ca²⁺]_i are not completely defined. To gain information in this regard, we examined the effects of low Na⁺ (35 mM) on freshly isolated cardiomyocytes from rat heart in the absence and presence of different interventions. The [Ca²⁺]_i in cardiomyocytes was measured fluorometrically with Fura-2 AM. Following a 10 min incubation, the low Na⁺-induced increase in [Ca²⁺]_i was only observed in cardiomyocytes depolarized with 30 mM KCl, but not in quiescent cardiomyocytes. In contrast, low Na⁺ did not alter the ATP-induced increase in [Ca²⁺]_i in the cardiomyocytes. This increase in [Ca²⁺]_i due to low Na⁺ and elevated KCl was dependent on the extracellular concentration of Ca²⁺ (0.25–2.0 mM). The L-type Ca²⁺-channel blockers, verapamil and diltiazem, at low concentrations (1 M) depressed the low Na⁺, KCl-induced increase in [Ca²⁺]_i without significantly affecting the response to low Na⁺ alone. The low Na⁺, high KCl-induced increase in [Ca²⁺]_i was attenuated by treatments of cardiomyocytes with high concentrations of both verapamil (5 and 10 M), and diltiazem (5 and 10 M) as well as with amiloride (5–20 M), nickel (1.25–5.0 mM), cyclopiazonic acid (25 and 50 M) and thapsigargin (10 and 20 M). On the other hand, this response was augmented by ouabain (1 and 2 mM) and unaltered by 5-(N-methyl-N-isobutyl) amiloride (5 and 10 M). These data suggest that in addition to the sarcolemmal Na⁺–Ca²⁺ exchanger, both sarcolemmal Na⁺–K⁺ATPase, as well as the sarcoplasmic reticulum Ca²⁺-pump play prominent roles in the low Na⁺-induced increase in [Ca²⁺]_i. (Mol Cell Biochem 263: 151–162, 2004)     

Relationship between Ca2+-transport and ATP hydrolytic activities in guinea-pig pancreatic acinar plasma membranes

Partially purified plasma membrane fractions were prepared from guinea-pig pancreatic acini. These membrane preparations were found to contain an ATP-dependent Ca²⁺-transporter as well as a heterogenous ATP-hydrolytic activity. The Ca²⁺-transporter showed high affinity for Ca²⁺ (K_Ca ²⁺ = 0.04 ± 0.01 M), an apparent requirement for Mg²⁺ and high substrate specificity. The major component of ATPase activity could be stimulated by either Ca²⁺ or Mg²⁺ but showed a low affinity for these cations. At low concentrations, Mg²⁺ appeared to inhibit the Ca²⁺-dependent ATPase activity expressed by these membranes. However, in the presence of high Mg²⁺ concentration (0.5–1 mM), a high affinity Ca²⁺-dependent ATPase activity was observed (K_Ca ²⁺ = 0.08 ± 0.02 M). The hydrolytic activity showed little specificity towards ATP. Neither the Ca²⁺-transport nor high affinity Ca²⁺-ATPase activity were stimulated by calmodulin. The results demonstrate, in addition to a low affinity Ca²⁺ (or Mg⁺)-ATPase activity, the presence of both a high affinity Ca²⁺-pump and high affinity Ca²⁺-dependent ATPase. However, the high affinity Ca²⁺-ATPase activity does not appear to be the biochemical expression of the Ca²⁺-pump.Abbreviations Ca²⁺-ATPase calcium-activated, magnesium-dependent adenosine triphosphatase - CaM calmodulin - CDTA trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetate - EDTA ethylene-diaminetetraacetate - EGTA ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetate - NADPH reduced form of nicotinamide adenine dinucleotide phosphate     

Stabilization of rat cardiac sacroplasmic reticulum Ca2+ uptake activity and isolation of vesicles with improved calcium uptake activity

Summmary The Ca²⁺ uptake activity of rat cardiac sacroplasmic reticulum (CSR) in ventricular homogenates is highly unstable, and this instability probably accounts for the low specific activity of Ca²⁺ uptake in previously reported fractions of isolated rat CSR. The instability was observed at either 0° or 37°, but the Ca²⁺ uptake activity was relatively stable at 25°. The decay of Ca²⁺ uptake activity at 0° could not be prevented by either PMSF or leupeptin, but dithiothreitol exerted some protective effects. Sodium metabisulfite prevented decay of the Ca²⁺ uptake activity of homogenates kept on ice but not of homogenates kept at 37°. We also found that release of the CSR from the cellular debris required homogenization in high KCI. This distinguishes rat CSR from canine CSR. Isolated CSR was produced by a combination of differential centrifugation and discontinuous sucrous gradient centrifugation. The average rate of the sustained oxalate-supported calcium uptake in the resulting CSR fraction was 0.36 mol/min-mg in the absence of CSR calcium channel blockers and 0.67 mol/min/mg in the presence of 10 M ruthenium red. Thus, this preparation has the advantage of containing both the releasing and non-releasing fractions of the CSR. The Ca²⁺-ATPase rates averaged 1.07 mol/min/mg and 0.88 mol/min-mg in the absence and presence of ruthenium red, respectively. Although these rates are higher than previously reported rates, this CSR preparation should still be considered a crude preparation. A major distinction between the rat CSR and dog CSR was the lower content of Ca²⁺-ATPase in rat CSR, as judged by SDS-PAGE. Preparations of CSR isolated by this method may be useful in evaluating alterations in CSR function.     

Calcium control of differentiation in Phytophthora palmivora

A method has been developed for the preparation of zoospores from Phytophthora palmivora which allows the ionic composition of the suspension medium to be closely controlled. Sub-micromolar concentrations of calcium ions have been shown to play a key role in maintaining the zoospore state and in the transition to the cyst stage. Restriction of free Ca²⁺ to between 0.2 and 1 M resulted in zoospores which could be maintained for several hours before they finally encysted and germinated. When exposed to citrus-pectin, or 3 mM SrCl₂, or to vigorous shaking, these zoospores underwent rapid synchronous encystment. At free Ca²⁺ concentrations below 0.1 M, zoospores lysed slowly. If exposed to inducers of encystment before lysis had occurred, the zoospores failed to respond to pectin or to vigorous shaking. However, they did differentiate in response to SrCl₂ addition. Provided the free Ca²⁺ was maintained between 0.02 and 0.2 M, zoospores survived gentle centrifugation, a procedure which previously had resulted in encystment.Abbreviations IM (ion-mix) release medium containing 100 M KCl, 10 M CaCl₂, and 10 M MgCl₂