The Ca2+-pumping ATPase and the major substrates of the cGMP-dependent protein kinase in smooth muscle sarcolemma are distinct entities

It has been proposed that the plasma membrane Ca2+ pump of smooth muscle tissues may be regulated by cGMP-dependent phosphorylation [Popescu, L. M., Panoiu, C., Hinescu, M. & Nutu, O. (1985) Eur. J. Pharmacol. 107, 393-394; Furukawa, K. & Nakamura, H. (1987) J. Biochem. (Tokyo) 101, 287-290]. This hypothesis has been tested on a smooth muscle sarcolemma preparation from pig thoracic aorta. The actomyosin-extracted membranes showed ATP-dependent Ca2+ uptake as well as cGMP-dependent protein kinase (G-kinase) activity. The molecular masses of the major protein substrates of the G-kinase (G1) and that of the Ca2+ pump were compared. Electrophoretic analysis of the phosphorylated intermediate of the sarcolemmal Ca2+-ATPase and the G1 phosphoprotein showed that these two proteins are not identical. The results were confirmed by using a 125I-calmodulin overlay technique and an antibody against human erythrocyte Ca2+-ATPase. Ca2+-uptake experiments with prephosphorylated membrane vesicles were carried out to elucidate possible effects of cGMP-dependent phosphorylation of membrane proteins on the activity of the Ca2+ pump. The cGMP-dependent phosphorylation was found to be extremely sensitive to temperature leading to very low steady-state phosphorylation levels at 37 degrees C. The difficulty was overcome by ATP[gamma S], which produced full and stable thiophosphorylation of G1 during the Ca2+-uptake experiments at 37 degrees C. However, the cGMP-dependent thiophosphorylation failed to influence the Ca2+-uptake properties of sarcolemmal vesicles. The results show that the Ca2+ pump of smooth muscle plasma membrane is not a direct target of the cGMP-dependent protein kinase and is not regulated by the cGMP-dependent phosphorylation of membrane proteins.     

The asymmetric effect of lanthanides on Na+-gradient-dependent Ca2+ transport in synaptic plasma membrane vesicles

Lanthanides (La3+, Pr3+ and Tb3+) inhibit Na+-gradient-dependent Ca2+ influx into synaptic plasma membrane vesicles. 50% inhibition is obtained by 7 microM lanthanide concentration. The inhibition of the Na+-gradient-dependent Ca2+ uptake exhibits competitive kinetic behaviour. The apparent Km of the Ca2+ influx is increased from 50 microM in the absence of lanthanides to 118 microM in the presence of La3+, 170 microM in the presence of Pr3+ and 130 microM in the presence of Tb3+. The maximal reaction velocity is not altered (8.35 nmol Ca2+ transported per mg protein per min in the absence of lanthanides and 8.16 nmol/mg per min in the presence of lanthanides). Lanthanides also inhibited Na+-gradient-dependent Ca2+ efflux from synaptic plasma membrane vesicles that were preloaded with Ca2+ in a Na+-gradient-dependent manner. Introduction of La3+ into the interior of the synaptic plasma membrane vesicles by rapid freezing of the vesicles in liquid N2 and slow thawing had no effect on either Na+-gradient-dependent Ca2+ influx or efflux. Synaptic plasma membrane vesicles can be preloaded with Ca2+ also in an ATP-dependent manner. This form of Ca2+ uptake is also inhibited by La3+ though at higher concentrations than the Na+-gradient-dependent Ca2+ uptake. Na+-gradient-dependent efflux from synaptic plasma membrane vesicles preloaded in an ATP-dependent fashion ('inside-out' vesicles) unlike efflux from synaptic plasma membrane vesicles preloaded in a Na+-gradient-dependent manner was not inhibited by La3+. These findings suggest that the inhibition by La3+ is manifested asymmetrically on both sides of the synaptic plasma membrane. Lanthanides are probably not transported via the Na+-Ca2+ exchanger since Tb3+ entry measured by fluorescence of Tb3+-dipicolinic acid complex formation occurred at high Tb3+ concentrations only (1.5 mM or above) and was not Na+-gradient dependent.     

Mn 2+-stimulatedATPase in rat brain

Divalent cation ATPases were prepared from rat brain synaptic vesicles, synaptosomal plasma membranes, and plasma membranes from the brain stem and sciatic nerve and tested for optimal stimulation by Mn2+, Mg2+, or Ca2+. ATPase in the synaptic vesicle subfraction was optimally stimulated by Mn2+. All plasma membrane preparations were optimally stimulated by Mg2+. Separate Mn2+ and Mg2+ ATPases could not be distinguished by either chemical inactivation or substrate preference criteria. Mn2+ stimulated ATPase in the micromolar range and it is suggested that Mn2+ interaction with ATPase may be of physiological and/or toxicological importance by being related to the cellular metabolism of this element.     

Effect of nitric oxide donors on Ca2+-uptake in the rat heart and liver mitochondria

The influence of NO donors, nitroglycerin (NG) and sodium nitroprusside (SNP), on Ca2+- uptake in rat heart and liver mitochondria is studied. It is shown that in vivo NG causes a rapid dose-dependent increase of Ca2+-uptake in rat heart mitochondria most pronounced at 0,5-1,0 mg/kg weight NG. This sharp increase of Ca2+-uptake is not accounted for by changes in membrane potential of mitochondria (deltapsim) because deltapsim is not influenced by less than 1,0 mg/kg NG, and moreover, decrease by approximately 30% is observed at 1,0-1,5 mg/kg NG. In vitro, on the contrary, a concentration-dependent decrease in Ca2+-uptake caused by NG as well as SNP is observed together with simultaneous decrease of deltapsim and concentration-dependent release of Ca2+ from mitochondria via Ca2+-uniporter as the result of partial depolarisation of mitochondrial inner membrane. The data obtained give an evidence that increase in Ca2+-uptake caused by NO donor in vivo takes place independently of changes in deltapsim and also is not resulted from a direct action of NO on Ca2+-uniporter. These observations allow us to suppose that activation of mitochondrial Ca2+-uptake in vivo and corresponding decrease in cytosolic Ca2+ concentration could be involved in vasodilatory action of nitric oxide.     

The action of alpha-adrenergic agonists on plasma-membrane calcium fluxes in perfused rat liver

The effect of alpha-adrenergic agonists on Ca2+ fluxes was examined in the perfused rat liver by using a combination of Ca2+-electrode and 45Ca2+-uptake techniques. We showed that net Ca2+ fluxes can be described by the activities of separate Ca2+-uptake and Ca2+-efflux components, and that alpha-adrenergic agonists modulate the activity of both components in a time-dependent manner. Under resting conditions, Ca2+-uptake and -efflux activities are balanced, resulting in Ca2+ cycling across the plasma membrane. The alpha-adrenergic agonists vasopressin and angiotensin, but not glucagon, stimulate the rate of both Ca2+ efflux and Ca2+ uptake. During the first 2-3 min of alpha-agonist administration the effect on the efflux component is the greater, the net effect being efflux of Ca2+ from the cell. After 3-4 min of phenylephrine treatment, net Ca2+ movements are essentially complete, however, the rate of Ca2+ cycling is significantly increased. After removal of the alpha-agonist a large stimulation of the rate of Ca2+ uptake leads to the net accumulation of Ca2+ by the cell. The potential role of these Ca2+ flux changes in the expression of alpha-adrenergic-agonist-mediated effects is discussed.     

High affinity Ca2+-stimulated Mg2+-dependent ATPase in rat brain synaptosomes, synaptic membranes, and microsomes

High affinity Ca2+-stimulated Mg2+-dependent ATPase activity of nerve ending particles (synaptosomes) from rat brain tissue appears to be associated primarily with isolated synaptic plasma membranes. The synaptic membrane (Ca2+ + Mg2+)-ATPase activity was found to exhibit strict dependence on Mg2+ for the presence of the activity, a high affinity for Ca2+ (K0.5 = 0.23 microM), and relatively high affinities for both Mg2+ and ATP (K0.5 = 6.0 microM for Mg2+ and KM = 18.9 microM for ATP). These kinetic constants were determined in incubation media that were buffered with the divalent cation chelator trans-cyclohexane-1,2-diamine-N,N,N',N'-tetraacetic acid. The enzyme activity was not inhibited by ouabain or oligomycin but was sensitive to low concentrations of vanadate. The microsomal membrane subfraction was the other brain subcellular fraction with a high affinity (Ca2+ + Mg2+)-ATPase activity which approximated that of the synaptic plasma membranes. The two membrane-related high affinity (Ca2+ + Mg2+)-ATPase activities could be distinguished on the basis of their differential sensitivity to vanadate at concentrations below 10 microM. Only the synaptic plasma membrane (Ca2+ + Mg2+)-ATPase was inhibited by 0.25-10 microM vanadate. The studies described here indicate the possible involvement of both the microsomal and the neuronal plasma membrane (Ca2+ + Mg2+)-ATPase in high affinity Ca2+ transport across membranes of brain neurons. In addition, they suggest a means by which the relative contributions of each transport system might be evaluated based on their differential sensitivity to inhibition by vanadate.     

硝苯吡啶对腹腔神经节突触传递的阻断作用

本文应用细胞内记录技术,观察了钙通道阻滞剂硝苯吡啶（nifedipine)对离体豚鼠腹腔神经节突触传递的影响,硝苯吡啶（0.1-10umol/L)不影响所检细胞的静息膜电位,膜电阻及细胞内刺激引起的动作电位,但能显著阻断N-型胆碱能的突触传递,并且这种作用可被低钙模拟、高钙拮抗,硝苯吡啶（10umol/L)也不影响突触后膜对乙酰胆碱（ACh)的敏感性;但在高钾克氏液中,能减少微小兴奋性突触后电位（mEPSPs)的频率;在低钙和高镁克氏液中,能减少量子含量,而对量子大小无影响。结果表明,治疗量的硝苯吡啶(0.1umol/L)通过阻滞突触前膜钙内流及ACh的量子性释放,产生突触阻断作用。这可能是硝苯吡啶降压机理的一个组成部分。     

The role of mitochondria in presynaptic calcium handling at a ribbon synapse

Mitochondria are thought to be important in clearing calcium from synaptic terminals. It is unclear, however, whether the principal role of mitochondria in pre-synaptic calcium handling is to take up Ca2+ directly or to fuel Ca2+ removal by other mechanisms. We used patch clamp techniques and fluorescence imaging to examine calcium clearance mechanisms, including mitochondrial uptake, in single synaptic terminals of retinal bipolar neurons. We found that extrusion through the ATP-dependent Ca2+ pump of the plasma membrane is the dominant form of Ca2+ removal in the synaptic terminal. Calcium uptake into mitochondria was sometimes evident with large Ca2+ loads but was consistently observed only when plasma membrane extrusion was inhibited. We conclude that mitochondria act primarily as an energy source in clearance of Ca2+ from bipolar cell synaptic terminals.     

Stimulatory effect of regucalcin on ATP-dependent calcium transport in rat liver plasma membranes

The effect of regucalcin, a calcium-binding protein isolated from rat liver cytoplasm, on ATP-dependent calcium transport in the plasma membrane vesicles of rat liver was investigated. (Ca2+-Mg2+)-ATPase activity in the liver plasma membranes was significantly increased by the presence of regucalcin (0.1-0.5 \sgmaelig;M) in the enzyme reaction mixture. This increase was completely inhibited by the presence of sulfhydryl group modifying reagent Nethylmaleimide (5.0 mM NEM) or digitonin (0.04%), which can solubilize the membranous lipids. When ATP-dependent calcium uptake by liver plasma membrane vesicles was measured by using 45CaCl2, the presence of regucalcin (0.1-0.5 \sgmaelig;M) in the reaction mixture caused a significant increase in the 45Ca2+ uptake. This increase was about 2-fold with 0.5 \sgmaelig;M regucalcin addition. An appreciable increase was seen by 5 min incubation with regucalcin addition. The regucalcin-enhanced ATP-dependent 45Ca2+ uptake by the plasma membrane vesicles was completely inhibited by the presence of NEM (5.0 mM) or digitonin (0.04%). These results demonstrate that regucalcin activates (Ca2+-Mg2+)-ATPase in the liver plasma membranes and that it can stimulate ATP-dependent calcium transport across the plasma membranes.     

Menadione-treated synaptosomes as a model for post-ischaemic neuronal damage.

Menadione bisulphite increased endogenous oxygen-radical production by rat brain synaptosomes, as indicated by H2O2 generation. Increased oxygen-radical production was also demonstrated in synaptosomes prepared from menadione-treated rats and synaptosomes reoxygenated after an anoxic insult. Acetylcholine synthesis de novo was inhibited in synaptosomes incubated with menadione in vitro, in synaptosomes prepared from menadione-treated animals in vivo, and in depolarized post-anoxic synaptosomes. Intrasynaptosomal free Ca2+ was increased by menadione in vitro (50 microM), but this increase was not due to stimulation of Ca2+ entry into the nerve terminals. Acetylcholine release was stimulated by menadione in vitro, possibly as a consequence of the elevated intrasynaptosomal Ca2+ content. The Ca2+ contents of synaptosomes prepared from menadione (10 mg/kg)-treated animals in vivo and synaptosomes reoxygenated after anoxia were unchanged. In synaptosomes prepared from menadione-treated animals, acetylcholine release was no longer significantly stimulated by K+, whereas it was unchanged from control (normoxic) values in synaptosomes reoxygenated after anoxia. None of these treatments caused any measurable damage to the synaptic plasma membrane (as judged by the release of lactate dehydrogenase), or to synaptosomal phospholipases (as judged by choline release from membrane phospholipids). Synaptosomes prepared from menadione-treated rats were found to be a good model for the study of post-anoxic damage to nerve-terminal function.     

An endogenous inhibitor protein of synaptic plasma membrane (Ca2+ + Mg2+)-ATPase

An inhibitor protein of synaptic plasma membrane (Ca2+ + Mg2+)-ATPase was purified to apparent homogeneity from rat cerebrum by a molecular weight cut followed by chromatography of cytosol proteins with molecular weights between 10 000 and 3500 on DEAE-Sephadex at pH 5.2. The inhibitor could be partially inactivated by proteinases and dithiothreitol, but was heat-stable. Gel filtration gave a molecular weight of about 6000. Like the (Ca2+ + Mg2+)-ATPase inhibitor protein isolated from erythrocytes, the inhibitor from brain contains a characteristic high proportion of glutamic acid (36%) and glycine (37%) residues. Synaptic plasma membrane Mg2+-ATPase and microsomal membrane (Ca2+ + Mg2+)-ATPase did not respond to the inhibitor. Synaptic plasma membrane and erythrocyte membrane (Ca2+ + Mg2+)-ATPases, however, were affected. Inhibitory influence on synaptic membrane (Ca2+ + Mg2+)-ATPase was reversible, since inhibition could be relieved upon removal of inhibitor from saturable sites on the membrane. The inhibitor is not a calmodulin-binding protein, since the concentration of calmodulin for half-maximal activation of the ATPase was unaffected by its presence. Mode of inhibition of the (Ca2+ + Mg2+)-ATPase by the inhibitor was non-competitive.     

The inhibitor of liver plasma membrane (Ca2+-Mg2+)-ATPase. Purification and identification as a mediator of glucagon action

Rat liver plasma membranes contain (Ca2+-Mg2+)-ATPase sensitive to inhibition by both glucagon and Mg2+. We have previously shown that Mg2+ inhibition is mediated by a 30,000-dalton inhibitor, originally identified as a membrane-bound protein. In fact, this inhibitor is also present in the 100,000 X g supernatant of the total liver homogenate. Its purification was achieved from this fraction by a combination of ammonium sulfate washing, gel filtration, and cationic exchange chromatography. N-Ethylmaleimide (NEM) treatment caused the inactivation of the purified inhibitor, which suggested that this protein possesses at least one NEM-sensitive sulfhydryl group essential for its activity. Treatment of the liver plasma membranes with NEM resulted in a 2- and 5-fold decrease in the affinity of the (Ca2+-Mg2+)-ATPase for glucagon and Mg2+, respectively, while the basal enzyme activity remained unchanged. This effect of NEM was concentration-, pH-, and time-dependent, optimal conditions being obtained by a 60-min treatment of plasma membranes with 50 mM NEM, at pH 7 and at 4 degrees C. The presence of 0.5 mM Mg2+ during NEM treatment of the plasma membranes prevented NEM inactivation. Reconstitution experiments showed that addition of the purified inhibitor to NEM-treated plasma membranes restored the inhibitions of the (Ca2+-Mg2+)-ATPase by both magnesium and glucagon. It is proposed that the (Ca2+-Mg2+)-ATPase inhibitor not only confers its sensitivity of the liver (Ca2+-Mg2+)-ATPase to Mg2+, but also mediates the inhibition of this system by glucagon.     

Calcium-dependent dissociation of synaptotagmin from synaptic SNARE complexes

The formation of the synaptic core (SNARE) complex constitutes a crucial step in synaptic vesicle fusion at the nerve terminal. The interaction of synaptotagmin I with this complex potentially provides a means of conferring Ca2+-dependent regulation of exocytosis. However, the subcellular compartments in which interactions occur and their modulation by Ca2+ influx remain obscure. Sodium dodecyl sulfate (SDS)-resistant core complexes, associated with synaptotagmin I, were enriched in rat brain fractions containing plasma membranes and docked synaptic vesicles. Depolarization of synaptosomes triggered [3H]GABA release and Ca2+-dependent dissociation of synaptotagmin from the core complex. In perforated synaptosomes, synaptotagmin dissociation was induced by Ca2+ (30-300 microM) but not Sr2+ (1 mM); it apparently required intact membrane bilayers but did not result in disassembly of trimeric SNARE complexes. Synaptotagmin was not associated with unstable v-SNARE/t-SNARE complexes, present in fractions containing synaptic vesicles and cytoplasm. These complexes acquired SDS resistance when N-ethylmaleimide-sensitive fusion protein (NSF) was inhibited with N-ethylmaleimide or adenosine 5'-O-(3-thiotriphosphate), suggesting that constitutive SNARE complex disassembly occurs in undocked synaptic vesicles. Our findings are consistent with models in which the Ca2+ triggered release of synaptotagmin precedes vesicle fusion. NSF may then dissociate ternary core complexes captured by endocytosis and recycle/prime individual SNARE proteins.     

The role of mitochondrial permeability transition pore in transmembrane Ca2+-exchange in mitochondria

Ca2+-uptake accompanied with mitochondrial permeability transition pore (MPTP) opening is studied in rat liver mitochondria. In conditions of MPTP opening, as well as in conditions of MPTP blockage by cyclosporine A (CsA), Ca2+-uptake in mitochondria is counterbalanced by proton efflux into incubation medium. Independent of MPTP opening, observed stoichiometry of this exchange is 1Ca2+ : 1H+. MPTP opening dramatically decreases Ca2+-uptake in mitochondria: from approximately 400 nmol/mg protein in the presence of CsA to approximately 80-100 nmol/mg protein due to the increased mitochondrial membrane permeability. In the absence of CsA Ca2+-uptake is accompanied by the insensitive to Ca2+-uniporter blocker, ruthenium red (RR), release of Ca2+ from mitochondria which corresponds to as well RR-insensitive, but sensitive to CsA uptake of H+ into mitochondrial matrix. This calcium-proton exchange resulting from MPTP opening is observed only when Ca2+ uptake into matrix exceeds some basal level. The data are consistent with an assumption that, contrary to Ca2+-uniporter, MPTP has its own proton conductance. MPTP opening provides exchange of Ca2+ between mitochondria and medium which is coupled to the counterflow of protons into matrix space. Obtained data elucidate the physiological role of MPTP as regulatory mechanism for control of Ca2+-uptake level and intramitochondrial pH.     

Effects of calcium channel blockers on insulin secretion and 45Ca(2+)-uptake of rat islets stimulated by glucose or K(+)-depolarization.

Two calcium channel antagonists, verapamil and nifedipine, have been used to explore the dependence of secretion on voltage-gated influx of calcium. Both antagonists were able to suppress the secretory response to K(+)-depolarization as well as the stimulation of 45Ca(2+)-uptake. However, they inhibited only partially the stimulation of both secretion and 45Ca(2+)-uptake. However, they inhibited only partially the stimulation of both secretion and 45Ca(2+)-uptake induced by glucose, alone or with palmitate. The stimulation of 45Ca(2+)-uptake by K(+)-depolarization, unlike that induced by glucose, was not sensitive to norepinephrine, starvation or fatty acid oxidation inhibitors. Therefore, it is suggested that glucose either modifies the properties of the voltage-dependent calcium channel and/or accelerates the exchange of a particular intracellular pool of calcium.     

Characterization of the cyclic 3',5'-nucleotide phosphodiesterase actitivty associated with synaptosomal plasma membranes and synaptic junctions.

Some characteristics of the cyclic 3',5'-nucleotide phosphodiesterase (phosphodiesterase) activity associated with the synaptosomal plasma membrane (synaptic membrane) and the synaptic junction fractions of rat brain are reported. Kinetic analysis revealed that only one type of phosphodiesterase activity, with a Km of 2.10 19(-4) M for cyclic AMP, is associated with both fractions. The specific activities of the phosphodiesterase in synaptic membranes and synaptic junctions have been estimated at 23.4 nmol/min per mg protein and 22.5 nmol/min per mg protein, respectively. The synaptic junction-associated activity undergoes a 30% stimulation by Ca2+ while no Ca2+ sensitivity of the synaptic membrane-associated activity could be detected. Cytochemical studies performed on the synaptic membrane fraction demonstrated a predominant localization of phosphodiesterase activity over postsynaptic densities, while dense deposits were sometimes observed over the synaptic cleft region.     

Stimulation of plasma membrane Ca2+-pump ATPase of vascular smooth muscle by cGMP-dependent protein kinase: Functional reconstitution with purified proteins

A 240-kDa protein isolated from porcine aortic smooth muscle as a substrate for cGMP-dependent protein kinase (cGMP kinase) whose phosphorylation was in a close association with stimulation of partially purified plasma membrane Ca2+-pump ATPase by the kinase was later shown to represent splicing variants of type 1 inositol 1,4,5-trisphosphate (IP3) receptor. To further clarify the role played by this protein in the stimulation of Ca2+-pump ATPase, it was attempted in the present study to specifically remove the protein by immunoprecipitation with an antibody specific to type 1 IP3 receptor. Contrary to expectation, stimulation of the ATPase by cGMP kinase was still observed after removal of the IP3 receptor. Furthermore, cGMP kinase stimulated a highly purified preparation of Ca2+-pump ATPase deprived of IP3 receptor when the concentrations of the ATPase were low enough (10-20 nM) to make it retain a monomeric form, while it did not produce stimulation when the concentration of the enzyme was increased to 40 nM at which the enzyme is known to take an oligomeric, fully activated form insensitive to activation by calmodulin. Heat-inactivated cGMP kinase and cGMP kinase without cGMP failed to stimulate the highly purified Ca2+-pump ATPase. In addition, type I but not type I cGMP kinase was found to stimulate the ATPase. The stimulation of Ca2+-pump ATPase by cGMP kinase occurs without any detectable phosphorylation of the ATPase. In conclusion, cGMP kinase can stimulate the plasma membrane Ca2+-pump ATPase when it is in a monomeric form without phosphorylating the Ca2+-pump ATPase and that of the two cGMP kinase isozymes found in the vascular smooth muscle, only type I cGMP kinase participates in the stimulation.     

Effects of calcium chelators, divalent cations and sulfhydryl reagents on calcium uptake and motility of bovine spermatozoa

Addition of 1 mM Ca/EGTA complex (1:1 ratio) to an incubation medium containing 1.5 mM Ca2+ produced a notable increase in the Ca2+ cycling in ejaculated bovine spermatozoa. Similar results were also obtained with the Ca/EDTA and Ca/EDTA complexes or with the heavy metal chelator DTPA (50 microM). Ba2+, Ni2+ or Co2+ added at 0.1 mM concentration abolished the stimulatory effect of the Ca/EGTA complex on Ca2+ cycling, whereas it did not affect the calcium movement in the absence of the calcium chelator complex. It is concluded that small amounts of these cations should be bound to the plasma membrane of bovine spermatozoa and inhibit the cellular calcium influx. 0.1 mM Cd2+ and NEM or 1 mM diamide produced a calcium efflux from the spermatozoa together with an inhibition of cellular motility and an increase in glutamate-oxaloacetate transaminase release. Conversely the impermeant sulfhydryl reagent mersalyl caused a net calcium efflux but did not alter the cellular motility nor the transaminase release. It is suggested that the permeant thiol reagents could decrease the spermatozoal mobility by impairing the mitochondrial ATP-synthesis.     

Changes in the nature of calcium transport systems on the porcine sperm plasma membrane during epididymal maturation.

Comparative studies of 45Ca(2+)-transport across the plasma membrane were performed using porcine caput, corpus and cauda epididymal sperm. The Ca(2+)-uptake is dependent on the presence of the substrates for respiration and is sensitive to verapamil. The Ca(2+)-efflux is mediated by both Na(+)-dependent and -independent systems. In the immature sperm in caput epididymis, Na(+)-independent efflux is predominant, but it is gradually replaced by Na(+)-dependent efflux during the epididymal transit. The net activity of Ca2+ accumulation into sperm increases with the epididymal maturation.     

Ca2+-dependent phosphatase and Ca2+-dependent ATPase activities in plasma membranes of eel gill epithelium--III. Stimulation of branchial high-affinity Ca2+-ATPase activity during prolactin-induced hypercalcemia in American eels

Infusions of ovine prolactin for 10 days induced hypercalcemia in unfed American eels, Anguilla rostrata LeSueur, that tentatively was related to stimulation of branchial Ca2+-uptake mechanisms. Analysis of ATPase activities in the plasma membranes of the branchial epithelium in prolactin treated eels showed a specific stimulation of high-affinity Ca2+-ATPase. The results of this study form further evidence that the high-affinity Ca2+-ATPase activity represents the Ca2+-pump of the branchial epithelium.