Amine Storage Granules Research Articles

Uptake and storage of catecholamines in rabbit brain after chronic reserpine treatment.

Abstract The brains of rabbits treated chronically with small doses of reserpine have been examined by the histochemical fluorescence method for dopamine, noradrenaline and 5-hydroxytryptamine. Measurements were made 4 and 24 hr after the last reserpine injection both in vivo and in vitro. Evidence has been obtained that the small functionally-important pool of amine can be directly visualized in the fluorescence microscope. It was found to be present intraneuronally, probably localized to amine storage granules. The degree of functional recovery was correlated with the recovery, as shown by the increased fluorescence, of a small intraneuronal pool of amine and with the ability of amine storage granules to take up monoamines.

The recovery of noradrenaline in adrenergic nerve terminals of the rat after reserpine treatment.

Abstract Tissue concentrations of endogenous noradrenaline in heart, submaxillary gland, and gastrocnemic muscle have been examined after one large dose of reserpine (10 mg/kg) to rats. After the initial depletion of the amine, the concentration started to rise between 24 and 36 h. For about one week thereafter the amine recovery proceeded comparatively fast, then the rate of the recovery slowed. Between the 4th and the 6th weeks there was a pronounced drop in the noradrenaline concentration in all three tissues, apparently beginning in the 4th week with a maximal decrease of about 20% in the 5th week after reserpine. Thereafter the concentrations increased to approach normal about 6 weeks after reserpine. These results are discussed in relation to the axonal down-transport of newly formed amine storage granules and to the life-span of these granules in the nerve terminals. The different parts of the noradrenaline recovery curve appeared to reflect the axonal down-flow of granules. A theoretical recovery curve was calculated, based on granular transport. This curve was similar to the observed recovery curve. The claim is made that the recovery of adrenergic function and noradrenaline levels after reserpine is due to a down-transport of newly formed, amine storage granules to the nerve terminals. There seems little need for the theory that the storage function reappears in old, reserpine-blocked granules, as a mechanism for noradrenaline recovery after a large dose of reserpine.

The transport and life-span of amine storage granules in bulbospinal noradrenaline neurons of the rat.

The transport and life-span of amine storage granules in bulbospinal noradrenaline neurons of the rat.

Recovery of noradrenaline in adrenergic axons of rat sciatic nerves after reserpine treatment.

Abstract The recovery of noradrenaline in adrenergic axons of the rat sciatic nerve after a single dose of reserpine (10 mg/kg i.p.) has been studied in unligated nerves and nerves ligated for 6 h. In unligated nerves the recovery at 24 h after reserpine was about 14% of normal. The noradrenaline content then slowly rose to reach about normal concentrations 6–7 days after reserpine injection. In nerves ligated 6 h before death, about 8·0 ng of noradrenaline accumulated proximal to the ligation in normal animals. At 6 and 12 h after reserpine about 4% of normal amounts of noradrenaline were found. Thereafter the amount of accumulated noradrenaline rapidly increased to about normal levels on day 2 after reserpine. At this time the content in unligated nerves was only about 45% of normal unligated nerve. On days 3–5 after reserpine, supranormal accumulations of noradrenaline were found (statistically highly significant), having a maximum at day 4 of about 145% of normal. At this time the noradrenaline content in unligated nerve was only about 80% of normal. The results may indicate an increased synthesis and increased rate of downtransport of amine storage granules during the early recovery phase after reserpine. This phenomenon may be part of a feed-back mechanism operating after depletion of the transmitter in the nerve terminals.

Recovery of noradrenaline levels after reserpine compared with the life-span of amine storage granules in rat and rabbit.

Recovery of noradrenaline levels after reserpine compared with the life-span of amine storage granules in rat and rabbit.

Correlation of the recovery of the granular uptake-storage mechanism and the nerve impulse induced release of ( 3 H)noradrenaline after reserpine.

Abstract Rats were treated intraperitoneally with 10 mg/kg reserpine. After various time intervals 1 μg/kg ± [3H]noradrenaline (3H-NA) was administered intravenously, and the uptake into subcellular fractions of the submaxillary gland was measured or, in some of the rats, the sympathetic chain of the neck was electrically stimulated with 10 impulses/s for 30 min. The release of 3H-NA and the contraction response of the lower eye-lid were measured. A striking parallel was observed between the recovery of the 3H-NA uptake into the amine storage particles and the nerve impulse-induced release of 3H-NA, and also the recovery of the functional response. The somewhat earlier recovery of the 3H-NA uptake into the coarse fraction might reflect the existence of another type of amine storage granule or might represent granules present near the nerve cell membrane. A possible increase in the turnover of the adrenergic transmitter during the period of recovery after reserpine is discussed.

Calcium and exocytosis.

Amine secretion from electropermeabilized bovine chromaffin cells and human platelets requires Ca2+ and MgATP. There appears to be little correlation between the pH or potential of the interior of the amine storage granules of the chromaffin cells and the Ca2+ sensitivity or extent of secretion. The Ca2+ sensitivities of secretion for both preparations are increased by activators of protein kinase C. In the platelet, thrombin also increases the Ca2+ sensitivity. The thrombin-induced response is further enhanced by micromolar levels of GTP. The non-hydrolysable analogue GTP gamma S also potentiates the Ca2+-dependent secretory response, but this effect is additive to that seen by thrombin rather than synergistic, as is the case with GTP. GTP gamma S inhibits catecholamine secretion from bovine chromaffin cells. In both preparations the effects of GTP gamma S are inhibited by 10 microM GTP, even though GTP concentrations up to 1 mM are without effect when added alone. These results are consistent with there being two sites of action for the guanine nucleotides, one at the level of the agonist receptor and activated by GTP or one of its breakdown products, and the other one activated by GTP gamma S--possibly at the level of protein kinase C itself.

May the alterations in lipid fluidity-mediated platelet hypersensitivity contribute to accelerated aging of platelets in diabetes mellitus?

Reduced platelet membrane lipid fluidity occurring in diabetes implies that platelet membrane receptors are more exposed to the external environment. This, in turn, may result in an increased sensitivity of platelets from diabetic humans to platelet aggregating agents. Platelet hypersensitivity (PH) may lead to augmented effects of agonists on the release of amine storage granules from the platelets of diabetic individuals. Aggregating and release-inducing agents decrease platelet density, which is assumed to be relevant to the aging processes. Thus it has become commonly accepted that platelet senescence or aging contributes to platelet density heterogeneity (PDH). Alterations of dynamic parameters of platelet membranes in diabetes may thus underlie the increased rate of aging or senescence of platelets in diabetic subjects.

Platelet function in Watanabe heritable hyperlipidemic rabbits. Decreased sensitivity to thromboxane A2.

The characteristics of platelets from seven 5-7-month-old homozygous Watanabe heritable hyperlipidemic (WHHL) rabbits (plasma cholesterol, 13.9 +/- 1.7 mM, mean +/- SEM) were compared with those of platelets from normocholesterolemic age/weight- and sex-matched control rabbits (plasma cholesterol, 2.2 +/- 0.3 mM). Whole-blood platelet count and platelet size and protein content were not different in the two groups of rabbits, and the platelets from the WHHL rabbits were not enriched in cholesterol as indicated by identical mean cholesterol:phospholipid molar ratios (C/P). Responses of washed platelets stimulated with various agonists were studied to determine the effects of the genetically determined hypercholesterolemia on the various pathways of platelet aggregation in the absence of plasma components. In platelets from WHHL rabbits compared with controls, aggregation induced by ADP (0.5-5 microM) did not differ; collagen-induced (0.25-1.5 micrograms/ml) responses (aggregation, secretion of carbon-14-labeled serotonin from the amine storage granules of prelabeled platelets, and thromboxane A2 [TxA2] formation) were significantly less extensive; with aspirin-treated platelets, aggregation and secretion of granule contents induced by the TxA2 mimetic U46619 (0.25-1 microM) were significantly less extensive; and thrombin-induced (0.005-0.1 unit/ml) responses of untreated platelets (aggregation, secretion of granule contents, and TxA2 formation) or aspirin-treated platelets (aggregation and secretion of granule contents) did not differ. These observations are in direct contrast with previous studies of platelets from rabbits with diet-induced hypercholesterolemia, in which responses to TxA2 and thrombin were enhanced. Platelets from WHHL rabbits are hyposensitive to aggregation induced by TxA2.

Reserpine-induced effects in the adrenergic neuron as studied with cytofluorimetric scanning

The anterograde axonal transport of norepinephrine (NE), dopamine-β-hydroxylase (DBH), neuropeptide Y (NPY) and tyrosine hydroxylase (TH) was studied in the rat sciatic nerve after reserpine (10 mg/kg). The histochemical method of Hillarp and Falck was used to study NE and the indirect immunofluorescence technique to study DBH-, NPY- and TH-immunoreactive material (IR), using antisera produced in rabbits. The rats were given reserpine 18 hr, or 1–7 days before sacrifice. Before perfusion fixation one nerve was dissected, frozen and freeze-dried for studies on NE, and the contralateral nerve was then processed for immunofluorescence. The amount of fluorescent material accumulated proximal to a 12 hr crush was quantified in longitudinal sections of the nerve using a cytofluorimetric scanning method. During the early phase after reserpine (18 and 25 hr) the amounts of accumulated NE were undetectable or very low, near control levels at 2 day, and overshooting to 160% of control at 4 d after reserpine. Accumulations of DBH-IR, NPY-IR and TH-IR were also depressed initially to 60–70% of control. DBH-IR and TH-IR thereafter increased to supranormal levels (140% of control) at day 4, while NPY-IR did not exhibit any overshooting but accumulated in the normal range at 2, 4, and 7 days. The results indicate that the amount of material transported distally early after reserpine is depressed. After local vinblastine-treatment of the lumbar sympathetic ganglia the amounts of DBH-IR and TH-IR in the perikarya were markedly lower in reserpine treated rats than in controls, probably due to a decreased perikaryal synthesis of these two enzymes. Rectal temperature in these rats decreased during the initial 10 hr after reserpine by up to 3°C. Thus, the decreased synthesis may be caused by the lowered body temperature, which also may slow down the rate of anterograde axonal transport. The later overshooting in accumulated amounts of NE, DBH-IR an TH-IR gives support to the hypothesis that amine granules containing DBH and NE are produced and transported in supranormal amounts around the 4th day after reserpine. Also, the results indicate that a considerable fraction of TH-IR is transported with organelles, probably amine storage granules, in adrenergic axons. NPY, shown to be localized in amine storage granules, did not overshoot at day 4 after reserpine, in contrast to DBH-IR, TH-IR and NE. We suggest that the induction in perikaryal synthesis, triggered by reserpine, is restricted to mRNA coding for DBH and TH, while, mRNA coding for NPY is not induced. This may imply an altered biochemical composition (less NPY/DBH) of the amine granules in transit distally around day 4 after reserpine in rat adrenergic axons.

Relative lack of ATP-driven H+ translocase activity in isolated parotid secretory granules.

The possible presence of ATP-driven H+ translocase activity in isolated rat parotid secretory granules has been examined by several approaches. First the transmembrane pH difference measured by either [14C] methylamine or [3H]acetate distribution is not substantially affected by ATP in the presence of membrane-permeating anions. Second, despite a low intrinsic H+ permeability of parotid granule membranes, only a small variably detectable inside-positive transmembrane potential is observed (by altered distribution of radioactive ions) when ATP is added in the absence of permeant anions. Third, ATP-induced lysis of parotid granules is minor and appears to be independent of ATP hydrolysis. Finally, ATP-hydrolase activity of the parotid granule fraction is not stimulated by an H+ ionophore, nor is it susceptible to inhibition by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole at a concentration which decreases the measured ATPase of purified chromaffin granule membranes by more than 80%. These findings suggest that this exocrine secretory granule type, which is characterized by storage of a heterogeneous mixture of secretory proteins, exhibits H+ pump activity which is at most a small fraction of that observed in biogenic amine storage granules of neural and endocrine tissues.

Effects on the Buoyant Density of Rabbit Platelets of ADP and Agents That Increase the Concentration of Cyclic AMP

Rabbit platelets were aggregated by adenosine diphosphate (ADP), allowed to deaggregate and then separated into density subpopulations by centrifugation through discontinuous Stractan density gradients. Although ADP causes little or no release of the contents of the amine storage granules of rabbit platelets, ADP caused a decrease in platelet density as compared with control platelets subjected to the same procedures except for exposure to ADP. The density change persisted for at least four hours. The apparent size of platelets stimulated with ADP increased initially, but returned to control values during a one-hour period. A similar decrease in platelet density was observed with an albumin density gradient. Under conditions in which aggregation did not occur in response to ADP with ethylenediaminetetraacetic acid (EDTA) in the medium, little or no decrease in platelet density was observed. Agglutination with polylysine did not change platelet density. Thus, not only agents such as thrombin and plasmin that cause the release of the contents of the platelet granules decrease platelet density, but ADP also has this effect. Platelets would be exposed to all of these stimuli during thromboembolic processes, and their effect on platelets may account for the decrease in platelet density observed previously in experiments with rabbits with indwelling aortic catheters. Agents that increase the concentration of cyclic AMP (cAMP) in platelets (PGE1, adenosine, dibutyryl cAMP, forskolin, and papaverine) also decreased platelet density. This effect persisted when the platelets were washed and resuspended in fresh medium and was also demonstrable in plasma. Platelet size was gradually increased by prostaglandin E1 (PGE1) which maintains platelets in a disc shape and does not cause the release of granule contents, indicating that the decrease in platelet density caused by PGE1 may be attributable to platelet swelling.

Effects on the buoyant density of rabbit platelets of ADP and agents that increase the concentration of cyclic AMP

Rabbit platelets were aggregated by adenosine diphosphate (ADP), allowed to deaggregate and then separated into density subpopulations by centrifugation through discontinuous Stractan density gradients. Although ADP causes little or no release of the contents of the amine storage granules of rabbit platelets, ADP caused a decrease in platelet density as compared with control platelets subjected to the same procedures except for exposure to ADP. The density change persisted for at least four hours. The apparent size of platelets stimulated with ADP increased initially, but returned to control values during a one-hour period. A similar decrease in platelet density was observed with an albumin density gradient. Under conditions in which aggregation did not occur in response to ADP with ethylenediaminetetraacetic acid (EDTA) in the medium, little or no decrease in platelet density was observed. Agglutination with polylysine did not change platelet density. Thus, not only agents such as thrombin and plasmin that cause the release of the contents of the platelet granules decrease platelet density, but ADP also has this effect. Platelets would be exposed to all of these stimuli during thromboembolic processes, and their effect on platelets may account for the decrease in platelet density observed previously in experiments with rabbits with indwelling aortic catheters. Agents that increase the concentration of cyclic AMP (cAMP) in platelets (PGE1, adenosine, dibutyryl cAMP, forskolin, and papaverine) also decreased platelet density. This effect persisted when the platelets were washed and resuspended in fresh medium and was also demonstrable in plasma. Platelet size was gradually increased by prostaglandin E1 (PGE1) which maintains platelets in a disc shape and does not cause the release of granule contents, indicating that the decrease in platelet density caused by PGE1 may be attributable to platelet swelling.

Effects of Plasmin on Rabbit Platelets

The effects of plasmin have been examined because platelets may be exposed to plasmin in vivo and treatment of platelets with plasmin shortens platelet survival. Rabbit plasmin was prepared by urokinase activation of plasminogen immobilized on lysine-Sepharose. Plasmin caused rabbit platelets to aggregate and release the contents of their amine storage granules, but aggregation was slower than in response to ADP or thrombin. EDTA, prostaglandin E1, or creatine phosphate/creatine phosphokinase were inhibitory, but indomethacin was not. Deaggregation did not occur when platelets had been aggregated by a concentration of plasmin that caused extensive release of granule contents. EDTA or prostaglandin E1 caused deaggregation. Low concentrations of ADP and plasmin acted synergistically in causing platelet aggregation. Plasmin decreased the amounts of platelet membrane glycoproteins that stained with periodic acid-Schiff reagent; glycoprotein I was more susceptible than glycoprotein II and III. Concentrations of plasmin that induced the release of amine storage granule contents also released PAS-staining granule glycoproteins. Platelets incubated with plasmin, washed and resuspended, were not aggregated by ADP, but were aggregated strongly by the combination of fibrinogen and ADP, and bound 125I-fibrinogen to a greater extent than untreated platelets. Platelets preincubated with a high concentration of plasmin were unresponsive to thrombin, but were sometimes aggregated by fibrinogen. Plasmin decreased the buoyant density and increased the median size of platelets. Thus plasmin, as well as ADP and thrombin, may contribute to the density shift observed in platelets from rabbits in which thrombosis and continuous vessel injury have been induced.

Effect of Amino Sugars on Platelet Aggregation and on Fibrinogen Binding

The amino sugars glucosamine, galactosamine and mannosamine (30 mM) inhibited aggregation of human or rabbit platelets induced by ADP, collagen, thrombin, PAF or high concentrations of sodium arachidonate. 125I-fibrinogen binding during ADP-induced aggregation, and release of amine storage granule contents were also inhibited. Increasing the calcium concentration of the suspending medium to 5 mM did not overcome the inhibitory effect on the release reaction. The amino sugars deaggregated rabbit platelets that had been aggregated by ADP, collagen or thrombin, but deaggregated human platelets readily only when ADP was used as the aggregating agent. Fibrinogen-induced aggregation of chymotrypsin-treated platelets was blocked by the amino sugars. They did not inhibit platelet adherence to a collagen-coated glass surface, nor affect release of granule contents from the adherent platelets. Aggregation and release induced by low concentrations of sodium arachidonate or the divalent cation ionophore A23187 were potentiated, indicating that the effects of the amino sugars on platelets are more complex than simple inhibition of the lectin-like activity that becomes available on the surface of platelets that have undergone the release reaction. One of the effects of the amino sugars, however, is interference with the binding of fibrinogen to platelets. The effects of the amino sugars are shared by other primary amines.

Noradrenaline and dopamine-beta-hydroxylase levels in rat salivary glands after preganglionic nerve stimulation: evidence for re-use of amine storage granules in transmitter release.

The levels of endogenous noradrenaline (NA) in rat salivary glands were reduced to about 40 per cent after 1 hour of preganglionic stimulation at 4 Hz. Following 2 to 3 hours of rest the NA values had recovered to normal and remained normal for 6 hours after the stimulation. The recovered NA could be depleted by renewed nerve stimulation. After treatment with reserpine the NA disappeared almost completely. The recovered NA is discussed to be mainly present in amine storage granules that are re-used for storage and transmitter release. The axonal down-transport of new granules to the nerve terminals appears to be too small, as based on earlier results, for being of any quantitative importance in the experimental situation. The dopamine-beta-hydroxylase (DBH) level in the glands did not significantly change after the period of stimulation. The DBH content remained mainly unchanged following either post-stimulation recovery, repeated stimulation, or reserpine treatment. First when the glands were stimulated for 4 hours, there was a small decrease of the DBH levels. The mainly unchanged levels of DBH after stimulation does not appear to be explained by "trapping" in the tissue of released DBH, or recovery of the DBH by a marked axonal down-transport of new granules, as based on earlier results. The results of the DBH estimations support the view that the granules are not consumed to any marked extent at nerve activity, but can be re-used for nerve transmission. Furthermore, the results are in agreement with lack of correlation between released NA and released DBH.

Arachidonate-Induced Fibrinogen Binding to Thrombin-Degranulated Rabbit Platelets is Independent of Released ADP

It has been established that fibrinogen binding occurs during adenosine diphosphate (ADP)-induced platelet aggregation, but studies of fibrinogen binding to platelets stimulated with arachidonate or most other aggregating agents are complicated by the release of platelet granule contents, which include both ADP and fibrinogen. Therefore. thrombin-degranulated rabbit platelets that have released more than 90% of their amine storage granule contents have been prepared for studies of the binding of 125I-fibrinogen during aggregation induced by agents that cause the release reaction with untreated platelets. To establish that thrombin–degranulated platelets are suitable for investigations of fibrinogen binding, the responses of these platelets to ADP were studied before other aggregating agents were used. The patterns of ADP-induced aggregation, deaggregation, fibrinogen binding and loss of bound fibrinogen, as well as the effects of inhibitors of platelet aggregation, were similar to those exhibited by untreated control platelets. The fibrinogen binding during ADP-induced aggregation was specific, saturable and reversible, and the apparent number of fibrinogen receptors and their association constant were in the same range as those calculated for control platelets. These thrombin-degranulated platelets have been used to study arachidonate-induced aggregation and 125l-fibrinogen binding. Responses to arachidonate were similar to ADP-induced responses except for the effects of creatine phosphate/creatine Phosphokinase (CP/CPK), which converts ADP to ATP, and of indomethacin, which prevents the conversion of arachidonate to prostaglandin endoperox-ides and thromboxane A2. CP/CPK abolished ADP-induced aggregation and fibrinogen binding of thrombin-degranulated platelets, but had much less effect on the responses to arachidonate. On the other hand, indomethacin had no effect on responses to ADP, but blocked aggregation and fibrinogen binding caused by arachidonate. We observed no evidence of synergism between arachidonate and the low residual concentration of ADP (less than 0.06 μM) that might have been released from the thrombin-degranulated platelets. Thus, arachidonate, through its products, appears to cause aggregation and fibrinogen binding to thrombin-degranulated platelets by a mechanism independent of ADP, although ADP may enhance the reactions. These products probably act by exposing fibrinogen receptors on the platelet surface in a manner similar to ADP.

The Use of the Synthetic Peptide, Gly-Pro-Arg-Pro, in the Preparation of Thrombin-degranulated Rabbit Platelets

The method for preparing thrombin-degranulated platelets has been modified to avoid the use of plasmin or successive treatments with small amounts of thrombin, while still achieving more than 90% release of platelet amine storage granule contents. It was necessary to prevent the fibrinogen released from the platelets during thrombin treatment from forming an insoluble fibrin mesh that could trap the platelets and hinder their deaggregation. To accomplish this we have treated rabbit platelets with 0.73 U/ml of thrombin for 1 min in the presence of the synthetic peptide, Gly-Pro-Arg-Pro, which prevents the polymerization of fibrin molecules. We have demonstrated that it also prevents 125I, initially added as 125I-fibrinogen, from associating with the platelets in a form that was not removed by centrifuging and washing during the preparation of thrombin-degranulated platelets, and we infer that products formed from the fibrinogen released from the platelets would also be prevented from associating with them. Thrombin-degranulated platelets prepared by this method have lost 92% of their granule contents and they can be washed and resuspended. These platelets aggregate normally upon stimulation with thrombin, adenosine diphosphate (ADP), or arachidonate. Thus, Gly-Pro-Arg-Pro is useful in preparing thrombin-degranulated platelets for studying platelet reactions without the complicating effects of released materials such a ADP and fibrinogen.

The use of the synthetic peptide, Gly-Pro-Arg-Pro, in the preparation of thrombin-degranulated rabbit platelets

The method for preparing thrombin-degranulated platelets has been modified to avoid the use of plasmin or successive treatments with small amounts of thrombin, while still achieving more than 90% release of platelet amine storage granule contents. It was necessary to prevent the fibrinogen released from the platelets during thrombin treatment from forming an insoluble fibrin mesh that could trap the platelets and hinder their deaggregation. To accomplish this we have treated rabbit platelets with 0.73 U/ml of thrombin for 1 min in the presence of the synthetic peptide, Gly-Pro-Arg-Pro, which prevents the polymerization of fibrin molecules. We have demonstrated that it also prevents 125I, initially added as 125I-fibrinogen, from associating with the platelets in a form that was not removed by centrifuging and washing during the preparation of thrombin-degranulated platelets, and we infer that products formed from the fibrinogen released from the platelets would also be prevented from associating with them. Thrombin-degranulated platelets prepared by this method have lost 92% of their granule contents and they can be washed and resuspended. These platelets aggregate normally upon stimulation with thrombin, adenosine diphosphate (ADP), or arachidonate. Thus, Gly-Pro-Arg-Pro is useful in preparing thrombin-degranulated platelets for studying platelet reactions without the complicating effects of released materials such a ADP and fibrinogen.

Agonist selectivity and second messenger concentration in Ca2+-mediated secretion.

Cells typically carry receptors for several excitatory and/or inhibitory agonists. In many cases the pattern of response observed is characteristic of the particular agonist used1–4. These differences in the nature of the responses cannot always be explained on the basis that the agonists use different intracellular second messengers3–5. For example, in the blood platelet, Ca2+ seems to be the second messenger6 for such excitatory agonists as ADP, adrenaline, thrombin, collagen and 11-epoxy-methanoprostaglandin H2 (U-46619). These agonists cause secretion of serotonin, ADP and ATP from the amine storage granules whereas secretion of acid hydrolases from the lysosomes has been reported to occur only on stimulation by higher concentrations of collagen and thrombin7–11. We have investigated this agonist selectivity and report here results suggesting that it is not related to cytosolic Ca2+ concentration.