Effects of Mg2+ and Ca2+ on Noradrenaline Release and Uptake in Adrenergic Nerve Granules in Different Media

Euler , U. S. V. and F. Lishajko . Effects of Mg²⁺ and Ca²⁺ on noradrenaline release and uptake in adrenergic nerve granules in different media. Acta physiol. scand. 1973. 89. 415–422. Uptake and release of noradrenaline (NA) from a suspension of storage granules from bovine splenic nerves was measured after incubation in various media and effects of added Mg²⁺ and Ca²⁺ observed. No significant differences in NA release rate were observed in different media when NA uptake was prevented by addition of potassium ferricyanide to the medium. Uptake was facilitated by phosphate ions 5–30 mM but counteracted by monovalent cations. In sucrose-phosphate media uptake of NA almost balanced spontaneous release. Mg²⁺ and Ca²⁺ had only a small action on NA release in the absence of NA uptake. In sucrose phosphate media Mg²⁺ facilitated uptake, whereas Ca²⁺ enhanced NA release. This action was associated with the formation of a calcium phosphate precipitate. No release or uptake of NA was observed during incubation at ice-water temperature, nor did Mg²⁺ or Ca²⁺ have any effect. The results tend to show that release and uptake of NA in storage granules are separate, temperature-dependent processes, influenced by mono- and divalent cations and phosphate ions.     

Adenosine Triphosphate Dependent Calcium Uptake by Subcellular Fractions from Bovine Neurohypophyses

Bovine neurohypophyses were fractionated by differential and density gradient ultracentrifugation and the Ca²⁺ uptake and ATPase activities in the microsomal, mitochondrial and secretory granule fractions were studied. The microsomal and mitochondrial fractions accumulated Ca²⁺ in the presence of ATP. The accumulation by the latter per mg protein was at least twice as large as by the former. This Ca²⁺ accumulation was accompanied by liberation of inorganic phosphate (P_i). In the presence of sodium azide (2 mM) Ca²⁺ uptake and P_i liberation were inhibited in the mitochondrial, but not in the microsomal fraction. Further studies of the microsomal fraction revealed that the ATP-dependent Ca²⁺ uptake and P_i liberation activities were temperature and pH-dependent and required Mg²⁺. Both activities were stimulated by very low concentrations of Ca²⁺ (1–10 μM) and were inhibited by EGTA (2 mM). N-ethyl-maleimide (2 mM) inhibited both the Ca²⁺ uptake and ATPase activities of the microsomal fraction. These results suggest the presence of a membrane ATPase that is stimulated by both Ca²⁺ and Mg²⁺. It is suggested that the observed Ca²⁺ uptake activities are involved in maintaining a low axoplasmic free Ca²⁺ concentration, thus playing an important role in the release mechanism of vasopressin by the neuro-secretory terminals.     

Magnesium dependent contraction of glycerinated smooth muscle

The effect of Mg²⁺ on the contraction of glycerinated smooth and skeletal muscles was examined. In the absence of Ca²⁺, glycerinated smooth muscle contracted slowly when exposed to 5 mM adenosine triphosphate (ATP) and Mg²⁺. The magnitude of this contraction depended on the concentration of Mg²⁺ in a range of 1–20 mM. When 20 mM Mg²⁺ was used with ATP, glycerinated smooth muscle contracted almost maximally in a Ca²⁺ free environment, and it did not contract further on subsequent addition of 0.1 mM of free Ca²⁺ (EGTA-CaEGTA buffer). In contrast, in skeletal muscle a Ca²⁺ dependent contraction could always be elicited in the presence of 1–20 mM Mg²⁺ and 5 mM ATP. Tension development in smooth muscle induced by Mg²⁺ and ATP was not due to a contamination by Ca²⁺ or the removal of a Ca²⁺-sensitive protein. In the present experimental conditions, glycerinated smooth muscle developed a Mg²⁺-dependent contraction in a Ca²⁺ free medium, but glycerinated skeletal muscle did not.     

Intracellular metabolism of adenosine 3′,5′-cyclic monophosphate and calcium inward current in perfused neurones of helix pomatia

The effects of intracellular administration of substances known to affect the cyclic AMP metabolism on the calcium inward currents were studied on the somatic membrane of intracellularly perfused snail neurons. During intracellular perfusion of a neurone with 130 mM Tris aspartate solution the calcium currents, unlike the sodium ones, gradually decreased. The rate of decrease was greatly dependent upon temperature. Addition to the intracellular perfusate of exogenous cyclic AMP together with ATP and Mg²⁺ ions prevented the decrease and partially or completely restored the initial value of calcium currents; afterwards their decline was re-established but at a much lower rate. Separate intra-cellular administration of each of the latter agents did not produce a noticeable restoring effect. The maximum rate and degree of restoration could be achieved at 10^?4 M cyclic AMP, 2mM ATP and 3 mM Mg²⁺. Cyclic GMP at the same concentrations as cyclic AMP did not affect the calcium currents. A similar effect to that produced by exogenous cyclic AMP was caused by fluoride anions (8 mM) added to the perfusate together with 2mM ATP and 3mM Mg²⁺. Increase of intracellular concentration of fluoride anions up to complete substitution of aspartate produced only transitory restoration of the calcium current followed by its depression.Addition of CaCl₂(10^?4–10^?3 M) to the perfusate resulted in a rapid acceleration of the calcium current decline. This process could be slowed down with intracellular introduction of the cyclic AMP phosphodiesterase inhibitor theophylline (2 mM). Introduction of cyclic AMP together with ATP and Mg²⁺ ions also prevented the blocking of calcium conductance by intracellular calcium ions.The conclusion is drawn that the functioning of the main portion of calcium ionic channel population in the somatic membrane is closely linked with the intracellular level of cyclic AMP, probably through the activity of cyclic AMP-dependent protein kinases. Lowering of the cyclic AMP level due to its washing out during intracellular perfusion, acceleration of cyclic AMP hydrolysis by activation of phosphodiesterase or termination of cyclic AMP synthesis by exhaustion of the adenylate cyclase activity leads to a rapid transition of calcium channels to a non-functioning state, probably because of progressive dephosphorylation of the channel-forming membrane proteins.     

ADP exerts a protective effect against rundown of the Ca2+ current in bovine chromaffin cells

In isolated chromaffin cells, the high-voltage-activated Ca²⁺ current, recorded using 5 mM Ca²⁺ as the divalent charge carrier, exhibits rundown within 10 min, which is delayed for 1 h at least by the addition of 1 mM adenosine 5-triphosphate (ATP) to the pipette medium. The mechanism of this stabilizing action of ATP has been examined. ATP action is dose dependent; the rundown process, which was delayed at concentrations below 0.4 mM, was totally abolished at higher concentrations. The requirement for ATP was shown to be quite strict: 2 mM inosine 5-triphosphate (ITP) could not replace ATP, whereas guanosine 5-triphosphate (GTP) could, but at higher concentrations. This effect of ATP was shown to require the presence of MgCl₂ and the liberation of a phosphate group since the ATP analogue 5-adenylyl-imidodiphosphate (AMP-PNP) could not act as a substitute for ATP, suggesting an action through either adenosine 5-diphosphate (ADP) or a phosphorylation step. ADP, in the presence of Mg²⁺ only, could replace ATP in the same concentration range. This effect was shown to be specific to ADP; it was maintained after blocking the pathways which convert ADP into ATP, and could not be mimicked by guanosine 5-diphosphate (GDP). Similarly, ATP and ADP effects were abolished at an increased internal Ca²⁺ concentration (pCa 6 instead of pCa 7.7, where pCa = –log₁₀[Ca²⁺]). Nevertheless, the presence of 1 mM Mg-ADP in the bathing solution did not prevent the rundown of the Ca²⁺ channels when going to the inside-out patch recording configuration. In conclusion, the stabilizing effect of ATP may be interpreted by a Mg²⁺-ADP binding site present on high-voltage-activated Ca²⁺ channels. A localization of such an ADP regulatory site on the L-type Ca²⁺ channel itself cannot be excluded, though with an additional requirement since Mg-ADP alone is not able to maintain the corresponding activity on excised patches.     

Localization of ATPase activity in dendritic spines of the cerebral cortex

Summary An ATPase activity has been demonstrated in dendritic spines of the adult rat cerebral cortex using cerium to capture inorganic phosphate that is liberated during the enzymatic hydrolysis of ATP. Small pieces of cerebral cortex were fixed and incubated in a standard incubation medium containing both Ca²⁺ and Mg²⁺ at pH 7.2; other modifications of the incubation medium are described below. Electron microscopic examination of the cerium phosphate reaction product showed an electron dense precipitate localized in the cytoplasm of the spine behind the postsynaptic density. Whereas the postsynaptic density, itself, is not reactive, dense reaction product is seen immediately underneath the postsynaptic density and extending into the subsynaptic web. Reaction product is also associated with membranous cisternae within the dendritic spine. The reaction occurred in the presence of Ca²⁺ and Mg²⁺ and either of these two ions alone. However, virtually no reaction product is seen when the tissue was incubated in a medium devoid of Ca²⁺ and Mg²⁺, or in a medium containing Mg²⁺ and EGTA, suggesting that trace Ca²⁺ is necessary, but not sufficient for the reaction. Addition of p-chloromereurobenzoate, which selectively blocks SH groups, inhibited the reaction in the presence of Ca²⁺ and Mg²⁺, or both of these ions. The effect of pH on the reaction was determined using a lead precipitation method. The reaction occurred at pH 9.2 in the presence of Ca²⁺ alone. In the presence of Mg²⁺ alone, the reaction product appeared somewhat reduced at this pH. The presence of an ATPase activity, which is dependent upon Ca²⁺ in dendritic spines where actin and actin-binding proteins have also been localized, suggests that this activity may be involved in the dynamics of cytoskeletal function leading to shape changes in dendritic spines and synapses, as seen with various physiological and behavioral paradigms.     

Effects of ATP and related compounds on the Ca-induced Ca release mechanism of theXenopus SR

The effects of ATP and related compounds on the Ca²⁺ release mechanism of the sarcoplasmic reticulum (SR) was studied by using skinned skeletal muscle fibers ofXenopus laevis. ATP evoked marked Ca²⁺ release at very low level of Mg²⁺. , -Methylene analogue of ATP was almost as effective as ATP, which suggests Ca²⁺ release evoked by ATP is elicited without ATP hydrolysis. ADP and AMP also evoked Ca²⁺ release from the SR, but the effect of them became gradually weaker than that of ATP as the number of phosphates decreased. CTP, UTP and ITP were less potent than ATP. Adenosine also evoked more effective Ca²⁺ release than inosine. The compounds with adenine base, therefore, seem to elicit more potent Ca²⁺ release than those which have the same number of phosphates but do not consist of adenine base. AMP and Ca²⁺ ion evoked Ca²⁺ release synergistically, and the Ca²⁺ release responses evoked by ATP and related compounds showed the same pharmacological characteristics as Ca-induced Ca release. So, these Ca²⁺ release responses are construed as the manifestation of the same mechanism as Ca-induced Ca release. Effective concentration range of ATP and the effect of pyrophosphate on Ca²⁺ release evoked by ATP suggest that neither the high affinity ATP catalytic site of (Ca²⁺+Mg²⁺) ATPase of the SR nor the low affinity ATP binding site, reported by Dupont (1977), is implicated in the enhancement of these Ca²⁺ release responses from the SR.     

Ionic currents in the somatic membrane of rat dorsal root ganglion neurons-II. Calcium currents

The experiments have shown that the introduction of cyclic adenosine monophosphate, adenosine 5'-triphosphate and Mg²⁺ ions into dialysed isolated dorsal root ganglion neurons of 5–8 day-old rats not only prevents the rapid decline of calcium inward currents during the course of dialysis but restores to a considerable extent the calcium conductance. Introduction of adenosine 5'-triphosphate and Mg²⁺ has a much weaker stabilizing effect. This finding made it possible to separate and to investigate in detail the calcium current (I_Ca) in the somatic membrane of all investigated neurons. The maximal amplitude of I_Ca was proportional to the concentration of Ca²⁺ ions in the extracellular solution between 2 and 14mM; with higher concentration a saturation effect was observed. Replacement of Ca²⁺ by Ba²⁺ caused about a two-fold increase in the maximal amplitude of inward currents. Addition of Co²⁺, Mn²⁺, verapamil and related substances blocked the calcium current. The activation kinetics of I_Ca could be approximated by a modified Hodgkin-Huxley equation using a square power of the m-variable. The activation time constantτ_m changed in the range from 16 to 1.8 ms with testing potential change from ?40 to +20 mV. The inactivation of I_Ca was extremely slow; the half value of steady-state inactivation was observed at holding potential about ?60 mV. The potential-dependent and kinetic characteristics of the calcium currents obtained on several neurons from adult rats were similar to those for neurons of new-born ones.It is concluded that the somatic membrane of the rat neurons has a system of electrically-operated selective calcium channels, the normal functioning of which is dependent on intracellular cyclic nucleotide metabolism.     

ATP suppresses activity of Ca2+ -activated K+ channels by Ca2+ chelation

Ca²⁺-activated maxi K⁺ channels were studied in inside-out patches from smooth muscle cells isolated from either porcine coronary arteries or guinea-pig urinary bladder. As described by Groschner et al. (Pflügers Arch 417:517, 1990), channel activity (NP _o) was stimulated by 3 M [Ca²⁺]_c (1 mM Ca-EGTA adjusted to a calculated pCa of 5.5) and was suppressed by the addition of 1 mM Na₂ATP. The following results suggest that suppression of NP _o by Na₂ATP is due to Ca²⁺ chelation and hence reduction of [Ca²⁺]_c and reduced Ca²⁺ activation of the channel. The effect was absent when Mg ATP was used instead of Na₂ATP. The effect was diminished by increasing the [EGTA] from 1 to 10 mM. The effect was absent when [Ca²⁺]_c was buffered with 10 mM HDTA (apparent pK _Ca 5.58) instead of EGTA (pK _Ca 6.8). A Ca²⁺-sensitive electrode system indicated that 1 mM Na₂ATP reduced [Ca²⁺]_c in 1 mM Ca-EGTA from 3 M to 1.4 M. Na₂ATP, Na₂GTP, Li₄AMP-PNP and NaADP reduced measured [Ca²⁺]_c in parallel with their suppression of NP _o. After the Na₂ATP-induced reduction of [Ca²⁺]_c was re-adjusted by adding either CaCl₂ or MgCl₂, the effect of Na₂ATP on NP _o disappeared. In vivo, intracellular [Mg²⁺] exceeds free [ATP^4–], hence ATP modulation of maxi K⁺ channels due to Ca²⁺ chelation is without biological relevance.     

The influence of calcium on morphology and histamine content of isolated intact rat mast cell granules

Histamine is released by “sequential exocytosis” in mast cells. The exocytosis involves fusion of the plasma membrane with the perigranular membrane and further fusions of adjacent perigranular membranes. To study a possible direct effect of Ca²⁺ on granule membrane fusions, mast cell granule suspensions were prepared from sonicated rat mast cells. With the sonication method used, more than 60% of the granules obtained were found to be homogeneous, electron dense and surrounded by a perigranular membrane, when observed in the electron microscope. These granules correspond to normal, histamine-containing granules found in untreated mast cells and are therefore named “intact” granules. The other granules were swollen, less electron dense and without a perigranular membrane. These “changed” granules are formed during the histamine release process. Aliquots of the granule suspension were incubated in 0.34 M sucrose buffered with 10 mM HEPES, pH 7.0, containing different concentrations of CaCl₂, MgCl₂ (10 mM, 1 mM, 100 μM, 10 μM) or NaCl (10 mM). Only with the highest concentration (10 mM) of Ca²⁺ or Mg²⁺ was it possible to visualize an apposition of the perigranular membranes of “intact”, normal granules. No elimination of the individual membrane structures could be observed at the place of membrane contact. Thus, we found no signs of membrane fusions. The histamine content was lower in the suspensions incubated with lower concentrations of these ions or with 10 mM NaCl. Ca²⁺ and Mg²⁺ in high concentrations seemed to stabilize the perigranular membranes instead of initiating histamine release. Therefore, changes in the Ca²⁺-ion concentration per se do not explain the membrane fusions seen in mast cells during “sequential exocytosis”.     

Effect of chloride on Ca2+ release from the sarcoplasmic reticulum of mechanically skinned skeletal muscle fibres

 The effect of intracellular Cl^– on Ca²⁺ release in mechanically skinned fibres of rat extensor digitorum longus (EDL) and toad iliofibularis muscles was examined under physiological conditions of myoplasmic [Mg²⁺] and [ATP] and sarcoplasmic reticulum (SR) Ca²⁺ loading. Both in rat and toad fibres, the presence of 20 mM Cl^–in the myoplasm increased Ca²⁺ leakage from the SR at pCa (i.e. –log₁₀ [Ca²⁺]) 6.7, but not at pCa 8. Ca²⁺ uptake was not significantly affected by the presence of Cl^–. This Ca²⁺-dependent effect of Cl^– on Ca²⁺ leakage was most likely due to a direct action on the ryanodine receptor/Ca²⁺ release channel, and could influence channel sensitivity and the resting [Ca²⁺] in muscle fibres in vivo. In contrast to this effect, acute addition of 20 mM Cl^– to the myoplasm caused a 40–50% reduction in Ca²⁺ release in response to a low caffeine concentration both in toad and rat fibres. One possible explanation for this latter effect is that the addition of Cl^– induces a potential across the SR (lumen negative) which might reduce Ca²⁺ release via several different mechanisms. Received: 20 October 1997 / Received after revision: 1 December 1997 / Accepted: 2 December 1997     

Reversal of α1-receptor mediated relaxation in intestinal smooth muscle

The α₁-receptor agonist phenylephrine relaxed longitudinal rabbit jejunal muscle contracted in vitro by low concentrations of barium ions (1 mM). When the Ba²⁺ concentration was increased to 10–15 mM the response to phenylephrine was a contraction, and at Ba²⁺ concentrations in between the high and low range this response was biphasic—a relaxation followed by a contractile phase. The α₂-receptor agonist clonidine did not affect the tone of the Ba²⁺ contracted preparation. When the muscle preparation was contracted by Sr²⁺ (1–20 mM) in the presence of Ca²⁺ (2.5 mM), phenylephrine relaxed it, and no contractile response to phenylephrine was observed. In the absence of extracellular Ca²⁺, 5 mM Ba²⁺ caused a contraction. Under these conditions phenylephrine had no effect on the tissue tone. When Ca²⁺ was added in a low concentration (0.2-2 mM), phenylephrine elicited a gradually increasing contractile response. At 5 mM Ca²⁺ the contractile response was replaced by the normal relaxation. The contractile response to phenylephrine in the presence of 5 mM Ba²⁺ and 2.5 mM Ca²⁺ was partially blocked by low concentrations of verapamil. In higher concentrations verapamil abolished the tissue tonus completely. The contractile response to phenylephrine in the presence of 5 mM Ba²⁺ and 2.5 mM Ca²⁺ could be reverted to the normal relaxation by the addition of 20 mM Mg²⁺. Increasing the K⁺ concentration from the normal 5.9 to 62.9 mM blocked the phenylephrine-induced relaxation. No contractile response to phenylephrine occurred. It is concluded that Ba²⁺ could reverse the response of α₁ receptor stimulation in rabbit jejunum from a relaxation to a contraction and that this contractile response was dependent on the presence of Ca²⁺.     

Effects of changes in extracellular potassium,magnesium and calcium concentration on synaptic transmission in area CA1 and the dentate gyrus of rat hippocampal slices

The dependence of stimulus-induced synaptic potentials on changes of extracellular ionic concentrations of potassium ([K⁺]_o 3, 5, 8 mM), magnesium ([Mg²⁺]_o 2, 4, 8 mM) and calcium [Ca²⁺]_o (2 mM and continuous lowering by washing with Ca²⁺-free solutions) was investigated in area CA1 and dentate gyrus of rat hippocampal slices. Field potentials (fps), [K⁺]_o and [Ca²⁺]_o were measured with double-barreled ion selective/reference microelectrodes. Paired pulse stimulation (interval 50-ms) was applied either to the lateral perforant path or to the Schaffer collaterals. Elevation of [K⁺]_o from 5 to 8 mM and of [Mg²⁺]_o from 2 to 8 mM depressed the rise of excitatory postsynaptic potentials, as well as the amplitude of population spikes. With elevation of [K⁺]_o, the effect was stronger in the dentate gyrus, while with elevation of [Mg²⁺]_o, the reduction was more pronounced in area CA1. During washout of Ca²⁺, synaptic potentials became reduced and finally depressed. The [Ca²⁺]_o at which synaptic transmission was blocked increased with higher [Mg²⁺]_o and decreased with a change of [K⁺]_o from 3 to 5 mM, whereas with an elevation of [K⁺]_o from 5 to 8 mM, it rose in area CA1 but was reduced in dentate gyrus. All ionic changes also affected frequency habituation and potentiation in paired pulse experimentes. In dentate gyrus, frequency habituation was reversed to frequency potentiation with moderate lowering of [Ca²⁺]_o and with elevation of [Mg²⁺]_o and [K⁺]_o. In contrast, in area CA1 frequency potentiation was reduced upon elevation of [K⁺]_o.     

Inhibition of ω-conotoxin-sensitive Ca2+ channel currents by internal Mg2+ in cultured rat cerebellar granule neurones

The effects of changing the intracellular concentrations of either free Mg²⁺ ions ([Mg²⁺]_i) or Mg²⁺-bound adenosine triphosphate ([Mg · ATP]_i) on Ca²⁺ channel currents were assessed in cultured rat cerebellar granule neurones using the whole-cell patch-clamp technique. Raising [Mg²⁺]_i from 0.06 mM to 1.0 mM inhibited Ca²⁺ channel currents by approximately 50%. The action of -conotoxin GVIA (-CgTX), a selective inhibitor of N-type Ca²⁺ channels was also investigated. With increasing [Mg²⁺]_i, the proportion of current irreversibly blocked by -CgTX was reduced, and was negligible (approximately 5 pA of current) in the presence of [Mg²⁺]_i values of 0.5 mM or greater. Block of the -CgTX-sensitive current accounted for the reduction in total current by concentrations of [Mg²⁺]_i to 0.5 mM. Raising [Mg²⁺]_i had no effect on the rate of decay of Ca²⁺ currents, but did produce a negative shift in current activation, possibly due to a non-specific interaction with negative surface charge. Altering [Mg · ATP]_i from 0.3 to 5.0 mM caused a twofold increase in the size of currents without affecting the proportion of current sensitive to -CgTX. [Mg²⁺]_i was also effective in inhibiting the Ca²⁺ channel current following potentiation by increasing [Mg · ATP]_i. These data suggest that -CgTX-sensitive current in these cells is selectively inhibited by internal Mg²⁺ whereas both -CgTX-sensitive and -resistant components of current are potentiated by internal Mg · ATP. The mechanism by which Mg²⁺ inhibits N-type channels is unclear, but may involve an open channel block.     

Caffeine-induced calcium release from isolated sarcoplasmic reticulum of rabbit skeletal muscle

The essential conditions for the Ca²⁺ releasing action of caffeine from isolated sarcoplasmic reticulum (SR) of rabbits were evaluated by an investigation into the effects of Ca²⁺, Mg²⁺, MgATP^2–, and ATP concentration, ionic strength, and degree of loading. The heavy fraction (4,500×g) of the reticulum was used. Except for the study on degree of loading, 0.2 mg protein·ml^–1 SR was loaded actively with 0.02 mM⁴⁵CaCl₂, resulting in >90 nmol·mg protein^–1 at steady state, and then the effects of various parameters with or without (control) caffeine were tested.It was found that (1) caffeine induces a transient, dosedependent release of Ca²⁺, (2) the absolute amount of Ca²⁺ released by caffeine increases with the Ca²⁺ load of the SR, (3) increasing the ionic strength () from 0.09 to 0.3 lowers the threshold concentration of caffeine, (4) the SR is refractory to a repeated challenge by a caffeine concentration causing maximal effect, (5) caffeine-induced Ca²⁺ release increases with increasing (a) external Ca²⁺ concentrations up to 5 M total Ca²⁺ (or 3 M free Ca²⁺) and (b) free ATP concentrations up to 0.45 mM, and (6) caffeine-induced Ca²⁺ release is not affected by changes of either the Mg²⁺ or the MgATP^2– concentration.     

Effects of Mg2+ on Ca2+ handling by the sarcoplasmic reticulum in skinned skeletal and cardiac muscle fibres

The influence of myoplasmic Mg²⁺ (0.05–10 mM) on Ca²⁺ accumulation (net Ca²⁺ flux) and Ca²⁺ uptake (pump-driven Ca²⁺ influx) by the intact sarcoplasmic reticulum (SR) was studied in skinned fibres from the toad iliofibularis muscle (twitch portion), rat extensor digitorum longus (EDL) muscle (fast twitch), rat soleus muscle (slow twitch) and rat cardiac trabeculae. Ca²⁺ accumulation was optimal between 1 and 3 mM Mg²⁺ in toad fibres and reached a plateau between 1 and 10 mM Mg²⁺ in the rat EDL fibres and between 3 and 10 mM Mg²⁺ in the rat cardiac fibres. In soleus fibres, optimal Ca²⁺ accumulation occurred at 10 mM Mg²⁺. The same trend was obtained with all preparations at 0.3 and 1 M Ca²⁺. Experiments with 2,5-di-(tert-butyl)-1,4-benzohydroquinone, a specific inhibitor of the Ca²⁺ pump, revealed a marked Ca²⁺ efflux from the SR of toad iliofibularis fibres in the presence of 0.2 M Ca²⁺ and 1 mM Mg²⁺. Further experiments indicated that the SR Ca²⁺ leak could be blocked by 10 M ruthenium red without affecting the SR Ca²⁺ pump and this allowed separation between SR Ca²⁺ uptake and SR Ca²⁺ accumulation. At 0.3 M Ca²⁺, Ca²⁺ uptake was optimal with 1 mM Mg²⁺ in the toad iliofibularis and rat EDL fibres and between 1 and 10 mM Mg²⁺ in the rat soleus and trabeculae preparations. At higher [Ca²⁺] (1 M), Ca²⁺ uptake was optimal with 1 mM Mg²⁺ in the iliofibularis fibres and between 1 and 3 mM Mg²⁺ in the EDL fibres. In the soleus and cardiac preparations Ca²⁺ uptake was optimal between 1 and 10 mM Mg²⁺. The results of this study demonstrate that SR Ca²⁺ accumulation is different from SR Ca²⁺ uptake and that these two important determinants of muscle function are differently affected by Mg²⁺ in different muscle fibre types.     

Multiple actions of zinc on transmitter release at mouse end-plates

In mouse diaphragm, the increase in frequency of mini end-plate potentials (f _mepp), by Ca²⁺ or Ba²⁺ in 20 mM K⁺, was reversibly inhibited by Zn²⁺ in a manner consistent with competition between Zn²⁺ and Ca²⁺ at a site which interacts with only one atom of Zn with an apparent dissociation constant (K_i′) of about 0.015 mM. Between 0.5 mM and 2 mM, Zn²⁺ caused a rapid and reversible dose-dependent increasef _mepp in 20 mM K⁺/0 Ca²⁺. Prolonged or repeated exposure to Zn²⁺ produced a slow increase inf _mepp followed by a decline, which once started, was not modified by of Zn²⁺. The time course was prolonged in raised Mg²⁺, bekanamycin, or in 5 mM K⁺ solution, and graded with Zn²⁺ concentration, but total numbers of MEPPs induced by 0.1 mM, 1 mM or 4 mM Zn²⁺ were not significantly different. Whenf _mepp fell it became insensitive to Ca²⁺, Ba²⁺, La³⁺ (in 20 mM K⁺), ethanol and raised osmotic pressure. Before complete block of responses to Ca²⁺, the Ca²⁺/f_mepp dose/response curve in 20 mM K⁺ was shifted to the right. These results indicate that Zn²⁺ enters the terminal via voltage-gated Ca²⁺ channels that interact in a complex way with these ions and then acts (a) as a partial agonist at sites where Ca²⁺ normally governs transmitter release, and (b) to produce irreversible changes in the nerve terminal, associated with a rise and subsequent fall off _mepp and loss of sensitivity of the release mechanism to Ca²⁺ and other agents.     

A new myofilament contraction model with ATP consumption for ventricular cell model

A new contraction model of cardiac muscle was developed by combining previously described biochemical and biophysical models. The biochemical component of the new contraction model represents events in the presence of Ca²⁺–crossbridge attachment and power stroke following inorganic phosphate release, detachment evoked by the replacement of ADP by ATP, ATP hydrolysis, and recovery stroke. The biophysical component focuses on Ca²⁺ activation and force (F _b) development assuming an equivalent crossbridge. The new model faithfully incorporates the major characteristics of the biochemical and biophysical models, such as F _b activation by transient Ca²⁺ ([Ca²⁺]–F _b), [Ca²⁺]–ATP hydrolysis relations, sarcomere length–F _b, and F _b recovery after jumps in length under the isometric mode and upon sarcomere shortening after a rapid release of mechanical load under the isotonic mode together with the load–velocity relationship. ATP consumption was obtained for all responses. When incorporated in a ventricular cell model, the contraction model was found to share approximately 60% of the total ATP usage in the cell model.     

Effects of magnesium on45Ca uptake and release at different sites in rabbit aortic smooth muscle

Effects of 1.5 mM Mg²⁺ on muscle tension and on⁴⁵Ca uptake and release at different sites in the rabbit aortic media-intimal layer were investigated. The sustained contraction induced by either 10^?6 M norepinephrine (NE) or 60 mM K⁺ was not affected by 1.5 mM Mg²⁺ in the presence of 1.5 mM Ca²⁺. However, the contractions elicited with NE or K⁺ in 0.03 mM Ca²⁺-containing solution were inhibited by 1.5 mM Mg²⁺ by 67% and 27%, respectively. Total⁴⁵Ca uptake measured in the presence of either 1.5 mM or 0.03 mM Ca²⁺ was not affected by 1.5 mM Mg²⁺. The rate of residual⁴⁵Ca uptake (⁴⁵Ca uptake followed by a wash in La³⁺-containing solution at low temperature) measured in the presence of 1.5 mM Ca²⁺ was slightly lower in the presence of 1.5 mM Mg²⁺. However, the increase in rate of residual⁴⁵Ca uptake induced by NE or the net increase in the residual⁴⁵Ca uptake induced by K⁺ was not decreased by 1.5 mM Mg²⁺. The residual⁴⁵Ca uptake measured in the presence of 0.03 mM Ca²⁺ was reduced to 64% and 24% of controls by addition of 1.5 mM Mg²⁺ or Sr²⁺, respectively. A part of the residual⁴⁵Ca was released by NE. Uptake of⁴⁵Ca at this NE-affected Ca²⁺ site did not take place in the presence of 1.5 mM Mg²⁺ when the Ca²⁺ concentration of the medium was 0.03 mM. However, this⁴⁵Ca uptake component was only partially inhibited when the Ca²⁺ concentration of the medium was 1.5 mM. The NE-induced increase in⁴⁵Ca efflux was not inhibited by 1.5 mM Mg²⁺. From these results, Mg²⁺ appears to be a weak antagonist for both Ca²⁺ entry into the vascular smooth muscle cell and Ca²⁺ binding at a high affinity intracellular site.     

Effects of vanadate, phosphate and 2,3-butanedione monoxime (BDM) on skinned molluscan catch muscle

The effects of orthovanadate (V_i), inorganic phosphate (P_i) and 2,3-butanedione monoxime (BDM) on tension, force transients and the catch state (passive tension maintenance) were investigated in saponin-skinned fibre bundles of the anterior byssus retractor muscle (ABRM) of the bivalve mollusc Mytilus edulis at pH 6.7. During maximal Ca²⁺ activation isometric force was depressed by V_i (0.03–10 mM), P_i (10 mM) and BDM (50 mM). Force transients following quick stretches (0.1–0.3% of fibre length) were accelerated substantially by 1 mM V_i, 10 mM P_i or 50 mM BDM. These compounds also accelerated force responses in experiments in which ATP was released rapidly from caged ATP by flash photolysis at both pCa 4.7 (force rise) and at pCa>8 (force decline). The effects on the catch state were investigated in two types of experiments: (1) Ca²⁺ removal after maximal Ca²⁺ activation and (2) rapid ATP release during high-force rigor at pCa>8. In both cases rapid relaxation was followed by slow relaxation (slower than 2% of initial force per min). This later slow relaxation (catch) was insensitive to V_i (1–10 mM), P_i (10 mM) and BDM (50 mM) but was accelerated by 0.12 mM cAMP. Complete relaxation to almost zero force was attained by changing pH from 6.7 to 7.7 (pCa>8). We conclude that catch depends on cAMP- and pH-sensitive structures linking the myofilaments and not on the force-generating actomyosin cross-bridges that are sensitive to V_i, P_i and BDM.