Histochemical and cytochemical demonstration of Ca++-ATPase activity in the stellate cells of the adenohypophysis of the guinea pig

The histo- and cytochemical localization of Ca++-ATPase activity in the adenohypophysis of the guinea pig was studied utilizing a newly developed method (Ando et al. 1981). An intense reaction was observed in the wall of the blood vessels and between non-secretory cells (stellate cells) and endocrine cells of the pars distalis. Under the electron microscope the Ca++-ATPase reaction product was located extracellularly in relation to the plasmalemma of the stellate cells. This reaction was dependent on Ca++ and the substrate, ATP, and reduced by the addition of 0,1 mM quercetin to the standard incubation medium. Preheating of the sections before incubation completely inhibited the enzyme activity. When Mg++ in different concentrations were substituted for Ca++ in the incubation medium the reaction was always reduced. Both Ca++ and Mg++ in the incubation medium also reduced the reaction. The plasmalemma of the endocrine cells contains no demonstrable amount of Ca++-ATPase activity. The function of the Ca++-ATPase activity is discussed in relation to the regulation of the extracellular Ca++ concentration which seems to be important with respect not only to the secretory process of the endocrine cells but also to the metabolism of the adenohypophysis.     

Relationship between plasma membrane Ca2+-ATPase activity and acrosome reaction in guinea pig sperm

The results obtained by biochemical measurement demonstrated for the first time that significant decrease of the plasma membrane Ca²⁺-ATPase activity occurred during capacitation and acrosome reaction of guinea pig sperm. Ethaorynic acid, one kind of Ca²⁺-ATPase antagonists, inhibited the plasma membrane Ca²⁺-ATPase activity, but calmodulin (50μg/mL) and trifluoperazine (200- 500μmol/L) did not, suggesting that calmodulin is not involved in ATP-driven Ca²⁺ efflux from sperm. However, calmodulin is involved in the control of Ca²⁺ influx. TFP, one kind of calmodulin antagonists, accelerated the acrosome reaction and Ca²⁺ uptake into sperm cells significantly. Ca²⁺-ATPase antagonists, quercetin, sodium orthovandate, furosemide and ethacrynic acid promoted the acrosome reaction, but inhibited Ca2+ uptake, which cannot be explained by their inhibitory effects on the plasma membrane Ca²⁺-ATPase activity. It is speculated that this phenomenon might be caused by simultaneous inhibitions of the activities of Ca²⁺-ATPase present in the plasma membrane, the outer acrosome membrane and the outer mitochondrion membrane resulting in Ca²⁺ accumulation in the cytoplasm, which in turn blocks further Ca2+ entry through some negative feedback mechanism(s). The inhibitory effect of Ca²⁺-ATPase antagonist on glycolytic activity may also be the reason for Ca²⁺ accumulation in cytoplasm and inhibition of Ca²⁺ uptake.     

Substrate specificity of the erythrocyte Ca2+-ATPase

In the absence of Mg²⁺, the observed activity of the erythrocyte plasma membrane Ca²⁺-ATPase is due to the hydrolysis of CaATP at a low rate. In the presence of Mg²⁺, the activity of the enzyme is much higher, but it is inhibited by high levels of free Mg²⁺. This inhibition appears to be due to competition of Mg²⁺ and Ca²⁺ for a site on the enzyme, rather than for ATP.     

Calcium transport and Ca2+-ATPase activity in ram spermatozoa plasma membrane vesicles

Plasma membrane vesicles, isolated from ejaculated ram sperm, were found to contain Ca²⁺-activated Mg²⁺-ATPase and Ca²⁺ transport activities. Membrane vesicles that were exposed to oxalate as a Ca²⁺-trapping agent accumulated Ca²⁺ in the presence of Mg²⁺ and ATP. The $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ for Ca²⁺ uptake was 33 nmol/mg protein per h, and the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values for Ca²⁺ and ATP were 2.5 μM and 45 μM, respectively. 1 μM of the Ca²⁺ ionophore A23187, added initially, completely inhibited net Ca²⁺ uptake and, if added later, caused the release of Ca²⁺ previously accumulated. A Ca²⁺-activated ATPase was present in the same membrane vesicles which had a $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of 1.5 μmol/mg protein per h at free Ca²⁺ concentration of 10 μM. This Ca²⁺-ATPase had $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values of 4.5 μM and 110 μM for Ca²⁺ and ATP, respectively. This kinetic parameter was similar to that observed for uptake of Ca²⁺ by the vesicles. The Ca²⁺-ATPase activity was insensitive to ouabain. Both Ca²⁺ transport and Ca²⁺-ATPase activity were inhibited by the flavonoid quercetin. Thus, ram spermatozoa plasma membranes have both a Ca²⁺ transport activity and a Ca²⁺-stimulated ATPase activity with similar substrate affinities and specificities and similar sensitivity to quercetin.     

Specificity of the metal-salt method for the localization of Ca2+-ATPase activity studied in rat adenohypophysis tissue in a model system of polyacrylamide gel films

Summary The histochemical method using the lead-salt technique for the detection of Ca²⁺-ATPase activity has been studied quantitatively in rat adenohypophyseal cell homogenates incorporated in polyacrylamide gel films using cytochemical conditions which were applied for the ultrastructural localization of this enzyme (El-sherif and Bácsy, 1988, 1989). Polyacrylamide gel films including cell homogenates were fixed in the presence or absence of calcium chloride, incubated for Ca²⁺-ATPase activity, and the mean integrated absorbance was determined. Omission of Ca²⁺ or levamisole from the incubation medium, as well as substitution of ATP by -glycerophosphate resulted in signIficantly decreased activity. Incubations with media containing AMP or ADP instead of ATP resulted in absorbance values not signIficantly different from background values obtained with substrate-free media. Maximum absorbance was obtained when 1% CaCl₂ was added to the fixative. Absorbance values increased with incubation time up to 45 min. Most of these data correlated well with our previous findings for the ultrastructural localization of Ca²⁺-ATPase activity in rat adenohypophysis and it can be concluded that the method is valid and specific.     

Short-time effects of neuroactive steroids on rat cortical Ca2+-ATPase activity

Recent experimental evidence indicates that some steroid hormones, apart from their well-documented genomic actions, could produce non-genomic rapid effects, and are potent modulators of the plasma membrane proteins, including voltage- and ligand-operated ion channels or G protein-coupled receptors. Neuroactive steroids, 17β-estradiol, testosterone, pregnenolone sulfate and dehydroepiandrosterone sulfate, after a short-time incubation directly modulated the activity of plasma membrane Ca²⁺-ATPase purified from synaptosomal membranes of rat cortex. The sulfate derivatives of dehydroepiandrosterone and pregnenolone applied at concentrations of 10^?11–10^?6 M, showed an inverted U-shape potency in the regulation of Ca²⁺-ATPase activity. At physiologically relevant concentrations (10^?8–10^?9 M) a maximal enhancement of the basal activity reached 200%. Testosterone (10^?11–10^?6 M) and 17β-estradiol (10^?12–10^?9 M) caused a dose-dependent increase in the hydrolytic ability of Ca²⁺-ATPase, and the activity with the highest concentration of steroids reached 470% and 200%, respectively. All examined steroids decreased the stimulatory effect of a naturally existing activator of the calcium pump, calmodulin. The present study strongly suggests that the plasma membrane calcium pump could be one of the possible membrane targets for a non-genomic neuroactive steroid action.     

Immunochemical and cytochemical studies of relaxin-containing cells in the guinea pig uterus

Uteri and ovaries from cycling, pregnant, and lactating guinea pigs were studied for immunolocalization of relaxin with the light microscope. Endometrial gland cells (EGC) from the same group of animals were examined in the electron microscope for the presence of secretory granules. Those EGC that exhibited high numbers of granules were stained either for relaxin with the protein A colloidal gold method or for carbohydrate with the thiocarbohydrazide technique. Relaxin was found in EGC from middle and late pregnant animals but was not detected in ovaries or uteri from cycling animals. While cytoplasmic granules were noted in most EGC from cycling animals examined, the number of granules was greatest in uteri from estrus and proestrus animals. Granules in EGC from estrus animals contained a carbohydrate-rich material but did not contain relaxin. Endometrial gland cells from animals in early to middle stages of pregnancy (days 15 and 30) contained limited numbers of granules, almost all of which contained carbohydrate. At day 45 of pregnancy, EGC containing many granules were noted. The majority of granules contained relaxin; however, a significant number of EGC contained carbohydrate-rich granules. Infrequently, EGC were noted that contained two populations of granules, and these two populations were assumed to be made up of relaxin-containing and carbohydrate-rich granules. EGC from animals on day 60 of pregnancy typically contained granules, and the majority of these contained relaxin. Carbohydrate-rich granules were observed in EGC of the day 60 animals but were smaller in diameter and were noted in much lower numbers than the relaxin-containing granules. Endometrial gland cells from lactating animals infrequently contained granules. These studies are consistent with the hypothesis that the uterus is the primary source of relaxin in the guinea pig and that relaxin plays an important role in pregnancy and parturition of this species. The observations implicate endometrial glands and their products in the physiology of the cycling animal as well as the pregnant and parturient animal.     

Ultrastructural cytochemical and autoradiographic demonstration of nonspecific esterase(s) in guinea pig basophils

We used ultrastructural autoradiographic and cytochemical methods to localize esterase activities in unstimulated guinea pig basophils and in basophils undergoing degranulation or recovery from degranulation. We used tritium-labeled diisopropylfluorophosphate (DFP) as a probe for serine enzymes and localized this probe by ultrastructural autoradiography to cytoplasmic granules of immature or mature unstimulated basophils, as well as to granules released by degranulating basophils. Ultrastructural cytochemistry using alpha naphthyl acetate (ANA) as substrate localized nonspecific esterase activity to extruded granules, either within the interiors of degranulation sacs or within granules completely separated from degranulating basophils. Extruded granules retained their esterase activity for as long as 24 hr after antigen-induced degranulation. The plasma membranes of unstimulated or degranulating basophils, as well as of basophils recovering from degranulation, displayed prominent cell surface ANA esterase ectoenzyme activity. Lipid bodies, organelles present in the cytoplasm of both control and recovering basophils, were also alpha naphthyl acetate esterase (ANAE)-positive. Thus, cytochemical and autoradiographic techniques localized esterase and/or [3H]-DFP-binding activities to cytoplasmic granules, lipid bodies, and cell surface of basophils, and these enzyme activities persisted during both degranulation and recovery from degranulation.     

The role of the sites for ATP of the Ca2+-ATPase from human red cell membranes during Ca2+-phosphatase activity

1. In the presence of ATP, the Ca²⁺ pump of human red cell membranes catalyzes the hydrolysis of $\text{p-}\text{nitrophenyl}$ phosphate. The requirement for ATP of the Ca²⁺-$\text{p-}\text{nitrophenylphosphatase}$ activity was studied in relation to the two classes of site for ATP that are apparent during Ca²⁺ -ATPase activity. 2. (a) The $\text{K}{}_{0.5}$ for ATP as activator of the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ extrapolated at 0 mM PNPP is equal to the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of the Ca²⁺ -ATPase. (b) PNPP competes with ATP and its effectiveness is the same regardless the nucleotide acts as the substrate of the Ca²⁺ -ATPase or as activator of the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$. 3. PNPP at the high-affinity site does not substitute for ATP as activator of the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$. 4. At ATP concentrations that almost saturate the high-affinity site, Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ activity increases as a function of PNPP along an S-shaped curve, while Ca²⁺ -ATPase activity is partially inhibited along a curve of the same shape and apparent affinity. The fraction of Ca²⁺ -ATPase activity which is inhibited by PNPP is that which results from occupation of the low-affinity site by ATP. 5. Activation of the Ca²⁺ -ATPase by ATP at the low-affinity site is associated with inhibition of the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ activity. Both phenomena take place with the same apparent affinity and along curves of the same shape. 6. Experimental results suggest that: (a) the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ activity depends on ATP at the high-affinity site; (b) PNPP is hydrolyzed at the low-affinity site; (c) Ca²⁺ -ATPase activity at the high-affinity size persists during Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ activity.     

Inhibition of calmodulin-activated Ca2+-ATPase by propranolol and nadolol

Propranolol, at concentrations ranging from 0.05 to 0.5 mM, inhibits the calmodulin-activated Ca²⁺-ATPase of human erythrocyte membranes. In the same concentration range it is without effect on the basal Ca²⁺-ATPase. The inhibition is competitive and appears to be due to membrane binding, rather than to combination with cytoplasmic calmodulin as is the case for phenothiazines. This effect of propranolol may explain its ability to open the calcium-gated potassium channel, and could also be related to its action as a β-adrenergic blocker. Nadolol, another β-adrenergic blocker, is also an inhibitor of calmodulin-activated Ca²⁺-ATPase.     

Ca2+-ATPase cytochemistry in the spinal cord microvasculature of prenatal rats

Summary Membrane-bound Ca²⁺-ATPase activity was localized cytochemically in the blood vessels of the spinal cord of rat embryos to obtain a better understanding of the membrane activities of vascular cells.The cytochemical method revealed a growth of the parenchymal vasculature. In the parenchyma, reaction product was dense over the entire plasma membrane of voluminous endothelial cells provided with large nuclei and enriched cytoplasmic organelles, suggesting that the endothelial cells may be of a vascular sprout. The parenchymal vessels with a wide lumen were frequently associated with pericytes, and the Ca²⁺-ATPase activity was diminished in intensity on the luminal surface of the flattened endothelial cells. On the other hand, the endothelium of extraparenchymal capillaries exhibited Ca²⁺-ATPase activity primarily on the luminal surface of the plasma membrane. Quercetin, a Ca²⁺-transporting ATPase inhibitor, considerably decreased the abluminal activity in the voluminous endothelial cells with slit-like vascular lumen and the luminal activity of functioning capillary endothelium as well. Thus, a dual activity of Ca²⁺-ATPase, postulating for the activities of Ca²⁺-transporting ATPase and ecto-ATPase, was closely correlated with the maturation processes of the capillary endothelium.     

骨骼肌内质网Ca2+泵转运Ca2+的结构基础

Ca²⁺泵(Ca²⁺-ATPase)是调节细胞内Ca²⁺浓度的重要蛋白质之一. Ca²⁺泵在转运Ca²⁺的过程中经历一系列构象变化. 其中,E1状态为外向的Ca²⁺高亲和状态,E2状态则为内向的Ca²⁺低亲和状态. 目前,骨骼肌内质网Ca²⁺泵转运Ca²⁺过程中的几个中间状态,包括E1-2Ca²⁺,E1-ATP,E1-P-ADP,E2-Pi和E2状态的三维晶体结构已经解析. 介绍这几种状态的晶体结构,并分析Ca²⁺泵在执行功能过程中结构与功能的关系.     

Calmodulin antagonists inhibit Ca2+ uptake of mitochondria of guinea pig peritoneal macrophages

The Ca²⁺ uptake of the mitochondria of guinea pig peritoneal macrophages was not stimulated by the addition of calmodulin. However, calmodulin antagonists, both phenotiazines and N-naphthalenesulfonamides, in low concentrations inhibited the Ca²⁺ uptake of the mitochondoria, as compared to the inhibition of the calmodulin-dependent stimulation of brain phosphodiesterase. These calmodulin antagonists appear to have severe side effects on active processes of the mitochondria and which are unrelated to the specific effect on calmodulin.     

ATP-dependent Ca2+ accumulation in intracellular membranes of guinea pig macrophages after saponin treatment

Ca²⁺ transport system in the intracellular membranes was studied by using saponin-treated macrophages of the guinea pig, in which the plasma membranes could be selectively destroyed. Saponin-treated macrophages could accumulate 3.1 nmoles $\text{Ca}{}^{\mathrm{2+}}\text{4 × 10}{}^6$ macrophages in the presence of Mg-ATP and sodium azide with an apparent affinity constant of 6 × 10⁶ M^?1. In the absence of sodium azide, the value of Ca²⁺ uptake of saponin-treated macrophages was 95 nmoles/4 × 10⁶ macrophages, and its affinity constant for Ca²⁺ was 3 × 10⁵ M^?1. Saponin-treated macrophages may be suitable for the studies of Ca²⁺ transport systems in the intracellular membranes.