Caffeoylquinic acids from antiplasmodial active extract of Xanthium cavanillesii fruits and their molecular modelling studies.

The antiplasmodial active extract of Xanthium cavanillesii contains 3,4-dicaffeoyl quinic acid (3,4-DCQA), 3,5-dicaffeoyl quinic acid (3,5-DCQA) and 1,3,5-tricaffeoyl quinic acid (1,3,5-TCQA). These results inspired us to investigate the interaction of these molecules with a promising validated target of Plasmodium, PfATP6 orthologue of mammalian Ca+2-ATPase. Models of this receptor were obtained through comparative modelling. Afterwards, molecular docking studies were used to identify possible interaction modes of these caffeoyl quinic derivatives on the binding site. The 1,3,5-TCQA had the best energy, but all of these had better energy than thapsigargin, a non-competitive inhibitor of the sarco/endoplasmatic reticulum Ca+2-ATPase (SERCA).

Identification of novel inhibitors of human SPCA2.

To identify specific inhibitors of the human secretary pathway Ca(2+)-ATPase 2 (hSPCA2), a recombinant hSPCA2 was expressed in Saccharomyces cerevisiae, and purified by Co(2+)-chelating chromatography. The isolated hSPCA2 catalyzed ATP hydrolysis in the presence of Ca(2+) ions. The Ca(2+) dissociation constant for ATPase activation was 25 nM. hSPCA2 activity was inhibited by thapsigargin, 2,2'-methylenebis(6-tert-butyl-p-cresol), and 4-octylphenol in the low-micromolar concentration range. Unexpectedly, the organic solvent wash from standard laboratory polypropylene microtubes showed strong inhibitory potency toward hSPCA2 activity. The extract was found to comprise mainly primary fatty acid amides (PFAAs) by NMR analysis. Individual PFAAs, especially oleamide and linoleamide, almost completely inhibited hSPCA2 activity with IC50 values of 7.5 µM and 3.8 µM, respectively.

Effects of Dangkwisoo­san, a traditional herbal medicine for treating pain and blood stagnation, on the pacemaker activities of cultured interstitial cells of Cajal.

The interstitial cells of Cajal (ICCs) are the pacemaker cells in the gastrointestinal (GI) tract. In the present study, the effects of Dangkwisoo­san (DS) on pacemaker potentials in cultured ICCs from the small intestine of the mouse were investigated. The whole­cell patch­clamp configuration was used to record pacemaker potentials from cultured ICCs and the increase in intracellular Ca2+ concentration ([Ca2+i) was analyzed in cultured ICCs using fura­2­acetoxymethyl ester. The generation of pacemaker potentials in the ICCs was observed. DS produced pacemaker depolarizations in a concentration dependent manner in current clamp mode. The 4­diphenylacetoxy­N­methyl­piperidine methiodide muscarinic M3 receptor antagonist inhibited DS­induced pacemaker depolarizations, whereas methoctramine, a muscarinic M2 receptor antagonist, did not. When guanosine 5'­[ß­thio] diphosphate (GDP­ß­S; 1 mM) was in the pipette solution, DS marginally induced pacemaker depolarizations, whereas low Na+ solution externally eliminated the generation of pacemaker potentials and inhibited the DS­induced pacemaker depolarizations. Additionally, the nonselective cation channel blocker, flufenamic acid, inhibited the DS­induced pacemaker depolarizations. Pretreatment with Ca2+­free solution and thapsigargin, a Ca2+­ATPase inhibitor in the endoplasmic reticulum, also eliminated the generation of pacemaker currents and suppressed the DS­induced pacemaker depolarizations. In addition, [Ca2+]i analysis revealed that DS increased [Ca2+]i. These results suggested that DS modulates pacemaker potentials through muscarinic M3 receptor activation in ICCs by G protein­dependent external and internal Ca2+ regulation and external Na+. Therefore, DS were observed to affect intestinal motility through ICCs.

A pollen coat-inducible autoinhibited Ca2+-ATPase expressed in stigmatic papilla cells is required for compatible pollination in the Brassicaceae.

In the Brassicaceae, intraspecific non-self pollen (compatible pollen) can germinate and grow into stigmatic papilla cells, while self-pollen or interspecific pollen is rejected at this stage. However, the mechanisms underlying this selective acceptance of compatible pollen remain unclear. Here, using a cell-impermeant calcium indicator, we showed that the compatible pollen coat contains signaling molecules that stimulate Ca(2+) export from the papilla cells. Transcriptome analyses of stigmas suggested that autoinhibited Ca(2+)-ATPase13 (ACA13) was induced after both compatible pollination and compatible pollen coat treatment. A complementation test using a yeast Saccharomyces cerevisiae strain lacking major Ca(2+) transport systems suggested that ACA13 indeed functions as an autoinhibited Ca(2+) transporter. ACA13 transcription increased in papilla cells and in transmitting tracts after pollination. ACA13 protein localized to the plasma membrane and to vesicles near the Golgi body and accumulated at the pollen tube penetration site after pollination. The stigma of a T-DNA insertion line of ACA13 exhibited reduced Ca(2+) export, as well as defects in compatible pollen germination and seed production. These findings suggest that stigmatic ACA13 functions in the export of Ca(2+) to the compatible pollen tube, which promotes successful fertilization.

Effect of total lens epithelial cell destruction on intraocular lens fixation in the human capsular bag.

PURPOSE: To evaluate the effect of complete destruction of lens epithelial cells (LECs) in the capsular bag on intraocular lens (IOL) stability. SETTING: School of Biological Sciences, University of East Anglia, Norwich, United Kingdom. DESIGN: Comparative evaluation. METHODS: An in vitro organ culture model using the bag-zonule-ciliary body complex isolated from fellow human donor eyes was prepared. A capsulorhexis and fiber extraction were performed, and an Acrysof IOL was implanted. Preparations were secured by pinning the ciliary body to a silicone ring and maintaining it in 6 mL Eagle minimum essential medium supplemented with 5% v/v fetal calf serum and 10 ng/mL transforming growth factor-ß2 for 3 weeks or more. One bag of each pair was treated with 1 µM thapsigargin to destroy all LECs. Observations of LEC growth were captured by phase-contrast microscopy, IOL stability by video microscopy, and endpoint analysis through scanning electron microscopy and immunocytochemistry. RESULTS: The LECs in control capsular bags migrated centrally, closing the bag and fixating the IOL between the anterior and posterior capsules, as seen clinically. These events were not observed in the thapsigargin-treated group. After a period of controlled orbital movement, the IOL in the control group stabilized quicker than in the treated bags. There was no IOL rotation in the bag; however, the IOLs in the treated group rocked with axial movement. CONCLUSIONS: The LECs appeared to aid stabilization of current IOL designs in the capsular bag. The results have clinical implications for IOL design and for strategies to prevent posterior capsule opacification. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.

Regioselective N1-alkylation of 3,4-dihydropyrimidine-2(1H)-ones: screening of their biological activities against Ca(2+)-ATPase.

A regioselective N1-alkylation of 3,4-dihydropyrimidin-2(1H)-ones using a very efficient mild base Cs(2)CO(3) and alkyl halides at room temperature has been reported. The selectivity of this methodology is excellent and the yields of the alkylated products are very good. Furthermore inhibitory action of both the 3,4-dihydropyrimidin-2(1H)-ones and the N1-alkylated derivatives were tested on Ca(2+)-ATPase, which revealed that the parent compounds can act as Ca(2+)-ATPase inhibitors whereas the N1-alkylated derivatives are inefficient for this purpose.

Concentration dependent effect of (-)-Epicatechin on Na(+) /K(+) -ATPase and Ca(2+) -ATPase inhibition induced by free radicals in hypertensive patients: comparison with L-ascorbic acid.

Although the antioxidant properties of flavonoids are well documented, it is still unclear whether these effects are dependent on radical scavenging or iron chelating activities. Oxidative stress, a state of excessive reactive oxygen species (ROS) activity, is associated with vascular disease conditions such as hypertension. Both the anti- and pro-oxidant effects of tea catechins have been implicated in the alterations of cellular functions that determine their chemoprotective and therapeutic potentials in health and diseases. The present study examined the concentration dependent (10(-7) to 10(-4) m) effects of (-)-epicatechin and L-ascorbic acid on Na(+) /K(+) -ATPase and Ca(2+) -ATPase activity in hypertensive patients and normal subjects. L-ascorbic acid has been used as a positive control to compare the effect of (-)-epicatechin. A significant (p < 0.0001) decrease in the activities of Na(+) /K(+) -ATPase and Ca(2+) -ATPase was observed in hypertensive patients compared with normal subjects. We report that (-)-epicatechin shows a significant (p < 0.001) dose-dependent protective effect against oxidative stress induced by tert-butyl hydroperoxide (t-BHP), which is manisfested as a decrease in the activity of erythrocyte Na(+) /K(+) -ATPase and Ca(2+) -ATPase, in hypertensive patients as well as normal subjects. The effect of L-ascorbic acid was also significant (p < 0.001) and was comparable with that of (-)-epicatechin.

Ca(2+) homeostasis, Ca(2+) signalling and somatodendritic vasopressin release in adult rat supraoptic nucleus neurones.

Multiple mechanisms that maintain Ca(2+) homeostasis and provide for Ca(2+) signalling operate in the somatas and neurohypophysial nerve terminals of supraoptic nucleus (SON) neurones. Here, we examined the Ca(2+) clearance mechanisms of SON neurones from adult rats by monitoring the effects of the selective inhibition of different Ca(2+) homeostatic molecules on cytosolic Ca(2+) ([Ca(2+)](i)) transients in isolated SON neurones. In addition, we measured somatodendritic vasopressin (AVP) release from intact SON tissue in an attempt to correlate it with [Ca(2+)](i) dynamics. When bathing the cells in a Na(+)-free extracellular solution, thapsigargin, cyclopiazonic acid (CPA), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), and the inhibitor of plasma membrane Ca(2+)-ATPase (PMCA), La(3+), all significantly slowed down the recovery of depolarisation (50 mM KCl)-induced [Ca(2+)](i) transients. The release of AVP was stimulated by 50 mM KCl, and the decline in the peptide release was slowed by Ca(2+) transport inhibitors. In contrast to previous reports, our results show that in the fully mature adult rats: (i) all four Ca(2+) homeostatic pathways, the Na(+)/Ca(2+) exchanger, the endoplasmic reticulum Ca(2+) pump, the plasmalemmal Ca(2+) pump and mitochondria, are complementary in actively clearing Ca(2+) from SON neurones; (ii) somatodendritic AVP release closely correlates with intracellular [Ca(2+)](i) dynamics; (iii) there is (are) Ca(2+) clearance mechanism(s) distinct from the four outlined above; and (iv) Ca(2+) homeostatic systems in the somatas of SON neurones differ from those expressed in their terminals.

A phenylpropanoid, a slovenolide, two sulphur-containing germacranes and Ca2+-ATPase inhibitors from Thapsia villosa.

A phenylpropanoid 1, a slovenolide 2, and two germacranes bearing a methylthiopropenoate moiety, 3 and 4, along with twenty known metabolites have been isolated from the roots of Thapsia villosa var. villosa L. The structures of two known phenylpropanoids 5 and 6 have been corrected. Compounds 7 and 8 showed activity as potential inhibitors of the sarco- and endoplasmic Ca(2+)-dependent ATPases (SERCA) pump. Compounds 9, 10 and 11 increased significantly the cytoplasmic free calcium concentration ([Ca(2+)](c)) in human platelets in a concentration-dependent manner.

Capacitative Ca2+ entry during Ca2+ undershoot in bovine airway smooth muscle.

In numerous cells, Ca2+ undershoot is commonly observed after withdrawing stimulus that release Ca2+ from intracellular stores. In airway smooth muscle (ASM), the fast intracellular Ca2+ concentration ([Ca2+]i) drop during undershoot is produced by sarcoplasmic reticulum (SR) reloading, but the mechanisms involved in the long lasting basal [Ca2+]i recovery are unknown. We investigated the post-caffeine Ca2+ undershoot recovery in ASM isolated cells from bovine trachea. [Ca2+]i determination was done by a ratiometric method by incubating cells with Fura-2/AM. After inducing a transient response, caffeine withdrawn generated a Ca2+ undershoot. SR-Ca2+ content during maximum undershoot drop was approximately 40% of SR caffeine-releasable Ca2+ (SR-Ca2+ load). Undershoot recovery rate increased in presence of cyclopiazonic acid (CPA, a SR-Ca2+ ATPase inhibitor), but SR-Ca2+ load was reduced. Genistein (a tyrosine kinase inhibitor) slowed down the Ca2+ undershoot drop and the SR-Ca2+ load but did not affect the undershoot recovery rate. Ni2+ (a capacitative Ca2+ inhibitor), but neither SKF-96365 (a passive Ca2+ entry inhibitor) nor econazole (a capacitative Ca2+ inhibitor in non-excitable cells), inhibited Ca2+ undershoot recovery and SR-Ca2+ load. Our data suggest that capacitative Ca2+ entry is involved in bovine ASM Ca2+ undershoot recovery, and that changes in Ca2+ undershoot have an impact on SR-Ca2+ loading which might affect in turn ASM excitability.

Changes in expression and activity of the secretory pathway Ca2+ ATPase 1 (SPCA1) in A7r5 vascular smooth muscle cells cultured at different glucose concentrations.

Diabetes mellitus-related vascular disease is often associated with both a dysregulation of Ca2+ homoeostasis and enhanced secretory activity in VSMCs (vascular smooth muscle cells). Here, we employ a commonly used rat cell line for VSMCs (A7r5 cells) to investigate the effects of glucose on the expression and activity of the SPCA1 (secretory pathway Ca2+-ATPase 1; also known as ATP2C1), which is a P-type Ca2+ pump located in the Golgi apparatus that plays a key role in the secretory pathway. Our results show that mRNA expression levels of SPCA1 are significantly increased in A7r5 cells cultured in high glucose (25.0 mM)-supplemented medium compared with normal glucose (5.55 mM)-supplemented medium. SPCA1 protein expression levels and thapsigargin-insensitive Ca2+-dependent ATPase activity were also consistent with a higher than normal expression level of SPCA1 in high-glucose-cultured A7r5 cells. Analysis of AVP (arginine-vasopressin)-induced cytosolic Ca2+ transients in A7r5 cells (after pre-treatment with thapsigargin) showed faster rise and decay phases in cells grown in high glucose medium compared with cells grown in normal glucose medium, supporting the observation of increased SPCA expression/activity. The significant levels of both Ca2+-ATPase activity and AVP-induced Ca2+ transients, in the presence of thapsigargin, indicate that SPCA must play a significant role in Ca2+ uptake within VSMCs. We therefore propose that, if such increases in SPCA expression and activity also occur in primary VSMCs, this may play a substantial role in the aetiology of diabetes mellitus-associated vascular disease, due to alterations in Ca2+ homoeostasis within the Golgi apparatus.

Antinociceptive effects of 14-membered cyclopeptide alkaloids.

The analgesic potential of six 14-membered-ring cyclopeptide alkaloids, namely, franganine (1), discarine B (2), scutianines B (3), C (4), and D (5), and adouetine X (6), have been investigated. Among the compounds tested, only franganine (1) and adouetine X (6) produced antinociceptive effects in a mouse model of acute pain, without inducing undesirable side effects. Furthermore, compound 6 also exhibited a pronounced analgesic effect in a chronic neuropathic pain model in mice. It has been found that adouetine X (6) can decrease the activities of Ca(2+)-ATPase and Na(+)/K(+)-ATPase in vitro. Thus, the present findings have demonstrated that adouetine X (6) is a promising analgesic agent.

Attenuation by methyl mercury and mercuric sulfide of pentobarbital induced hypnotic tolerance in mice through inhibition of ATPase activities and nitric oxide production in cerebral cortex.

This study is aimed at exploring the possible mechanism of hypnosis-enhancing effect of HgS or cinnabar (a traditional Chinese medicine containing more than 95% HgS) in mice treated with pentobarbital. We also examined whether the effect of HgS is different from that of the well-known methyl mercury (MeHg). After a short period (7 days) of oral administration to mice, a nontoxic dose (0.1 g/kg) of HgS not only significantly enhanced pentobarbital-induced hypnosis but also attenuated tolerance induction; while a higher dose (1 g/kg) of HgS or cinnabar exerted an almost irreversible enhancing effect on pentobarbital-hypnosis similar to that of MeHg (2 mg/kg) tested, which was still effective even after 10 or 35 days cessation of administration. To study comparatively the effects of different mercury forms from oral administration of MeHg and HgS on membrane ATPase activities of experimental mice, analysis of the Hg content in the cerebral cortex revealed that correlated with the decrease of Na(+)/K(+)-ATPase and Ca(2+)-ATPase activities. Furthermore, NO levels of blood but not that of cerebral cortex were also decreased by mercuric compounds. Although pentobarbital alone enhanced cytochrome p450-2C9 in time dependent manner, all of mercurial compounds tested had no such effect. All of these findings indicated that the mercurial compounds including cinnabar, HgS and MeHg exert a long-lasting enhancing hypnotic activity without affecting pentobarbital metabolism, which provides evidence-based sedative effect of cinnabar used in Chinese traditional medicine for more than 2,000 years. The nontoxic HgS dosing (0.1 g/kg/day) for consecutive 7 days is perhaps useful for delaying or preventing pentobarbital-tolerance.

Influence of polyphenolic compounds isolated from Rubus coreanum on catecholamine release in the rat adrenal medulla.

The aim of the present study was to investigate whether polyphenolic compounds isolated from wine brewed from Rubus coreanum MIQUEL (PCRC) may affect the release of catecholamine (CA) from the isolated perfused rat adrenal medulla, and to establish its mechanism of action. PCRC (20-180 microg/mL) perfused into an adrenal vein for 90 min dose- and time-dependently inhibited the CA secretory responses evoked by acetylcholine (ACh, 5.32 mM), high K+ (a direct membrane-depolarizer, 56 mM), DMPP (a selective neuronal nicotinic Nn receptor agonist, 100 microM) and McN-A-343 (a selective muscarinic M1 receptor agonist, 100 microM). Also, in the presence of PCRC (60 microg/mL), the secretory responses of CA evoked by Bay-K-8644 (a L-type dihydropyridine Ca2+ channel activator, 10 microM), and cyclopiazonic acid (a cytoplasmic Ca2+-ATPase inhibitor, 10 microM) were significantly reduced, respectively. In the simultaneous presence of PCRC (60 microg/mL) and L-NAME (an inhibitor of NO synthase, 30 microM), the inhibitory responses of PCRC on the CA secretion evoked by ACh, high K+, DMPP, and Bay-K-8644 were considerably recovered to the extent of the corresponding control secretion compared with the inhibitory effect of PCRC alone. Taken together, these results obtained from the present study demonstrate that PCRC inhibits the CA secretory responses from the isolated perfused adrenal gland of the normotensive rats evoked by stimulation of cholinergic (both muscarinic and nicotinic) receptors as well as by direct membrane-depolarization. It seems that this inhibitory effect of PCRC is exerted by inhibiting both the calcium influx into the rat adrenal medullary chromaffin cells and the uptake of Ca2+ into the cytoplasmic calcium store partly through the increased NO production due to the activation of nitric oxide synthase (NOS), which are at least relevant to the direct interaction with the nicotinic receptor itself. It is also thought that PCRC might be effective in prevention of cardiovascular disease.

Effective protection of Terminalia catappa L. leaves from damage induced by carbon tetrachloride in liver mitochondria.

The protective effects of chloroform extracts of Terminalia catappa L. leaves (TCCE) on carbon tetrachloride (CCl4)-induced liver damage and the possible mechanisms involved in the protection were investigated in mice. We found that increases in the activity of serum aspartate aminotransferase and alanine aminotransferase and the level of liver lipid peroxidation (2.0-fold, 5.7-fold and 2.8-fold) induced by CCl4 were significantly inhibited by oral pretreatment with 20, 50 or 100 mg/kg of TCCE. Morphological observation further confirmed the hepatoprotective effects of TCCE. In addition, the disruption of mitochondrial membrane potential (14.8%), intramitochondrial Ca2+ overload (2.1-fold) and suppression of mitochondrial Ca2+-ATPase activity (42.0%) in the liver of CCl4-insulted mice were effectively prevented by pretreatment with TCCE. It can be concluded that TCCE have protective activities against liver mitochondrial damage induced by CCl4, which suggests a new mechanism of the hepatoprotective effects of TCCE.

Effect of maternal manganese blood levels on erythrocyte calcium-pump activity in newborns.

UNLABELLED: Manganese (Mn) is widely distributed in the biosphere but occurs in only trace amounts in animal tissues. Although Mn deficiency and toxicity both have pathological consequences, the underlying biochemical lesions have not been well defined. In vitro studies suggest that transport proteins are affected by Mn, lead (Pb), and selenium (Se). Among these transport proteins, the calmodulin-regulated calcium pump (Ca(2+)Mg(2+)ATPase) could be inhibited by Mn. In order to understand Mn biochemical pathways, we examined the relationships between Mn blood levels and red blood cell Ca-pump activity among 248 mothers and newborns, environmentally exposed to Mn, Pb, and Se. POPULATION AND METHODS: 248 mother-newborn pairs were recruited at Robert Debré University Hospital (Paris). Blood Mn and Pb concentrations were measured by absorption spectrophotometry. Se was measured by fluorometric method. Red blood cell membrane suspensions were obtained for Ca-pump activity measurements. Linear and quadratic regression models and Pearson correlation were performed. RESULTS: A non-linear parabolic relationship between maternal Mn blood levels and newborn Ca-pump activity was discovered from the analysis of the observed data. The peak level of maternal Mn that corresponded to a maximal activity of the newborn Ca-pump was estimated at 23.9 microg/l with a 95% confidence interval of 17.6 to 32.4 microg/l. An inhibition of this enzyme was observed at low and high levels of maternal Mn. The relationships between the newborn Ca-pump activity and maternal Se and Pb levels became non-significant after adjustment on all the co-factors included in the final model. CONCLUSION: Maternal environmental exposure to Mn, as reflected by maternal blood levels of this metal, is associated with a reduced activity of newborn erythrocyte Ca-pump in a non-linear pattern. Mn levels between 17.6 and 32.4 microg/l in maternal blood probably correspond to the optimal physiological concentration for the metabolism of this enzyme in newborns.

SERCA-inhibiting activity of C-19 terpenolides from Thapsia garganica and their possible biogenesis.

An investigation of Thapsia garganica afforded a series of tetracyclic C-19 dilactones, whose production was dependent on the time and location of the collection. These unusual tetrahomosesquiterpenoids are presumably biosynthesized via a carbon dioxide-triggered electrophilic polyolefin cyclization. Despite the structural differences with thapsigargin, these compounds showed SERCA-inhibiting properties.

Regulation of Ca2+ movements by cyclovirobuxine D in ECV304 endothelial cells.

In Fura-2/loaded ECV304 endothelial cells cyclovirobuxine D promoted a transient increase in cytosolic free Ca2+ originating from both an intracellular pool sensitive to the endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin and the extracellular space. The intracellular pool was apparently different from that mobilized by other agents acting through IP3 generation. The integrity of the plasma membrane was an absolute requirement. In cells treated with digitonin, cyclovirobuxine D did not promote any Ca2+ release from the intracellular stores even at high concentrations and in the absence or presence of thapsigargin or sodium azide, the inhibitors of the endoplasmic reticular or mitochondrial Ca2+ uptake. Furthermore, cyclovirobuxine D was effective in halting the persistent increase in cytosolic Ca2+ caused by thapsigargin, inhibiting the operation of the "capacitative" Ca2+ membrane channels as demonstrated by the decrease in the extent of both Ca2+-overshoot and Mn2+ influx. Additional effects of cyclovirobuxine D included a depolarization of plasma membrane apparently related to an enhanced influx of Na+ from the extracellular space. The results obtained indicate that cyclovirobuxine D markedly affects intracellular Ca2+ homeostasis in ECV304 endothelial cells by both promoting a discharge of intracellular pools and by interfering with the operation of store-dependent channels via plasma membrane depolarization.

Physiological characterization of 45Ca2+ and 65Zn2+ transport by lobster hepatopancreatic endoplasmic reticulum.

The crustacean hepatopancreas is an epithelial-lined, multifunctional organ that, among other activities, regulates the flow of calcium into and out of the animal's body throughout the life cycle. Transepithelial calcium flow across this epithelial cell layer occurs by the combination of calcium channels and cation exchangers at the apical pole of the cell and by an ATP-dependent, calcium ATPase in conjunction with a calcium channel and an Na+/Ca2+ antiporter in the basolateral cell region. The roles of intracellular organelles such as mitochondria, lysosomes, and endoplasmic reticulum (ER) in transepithelial calcium transport or in transient calcium sequestration are unclear, but may be involved in transferring cytosolic calcium from one cell pole to the other. The ER membrane has a complement of ATP-dependent calcium ATPases (SERCA) and calcium channels that regulate the uptake and possible transfer of calcium through this organelle during periods of intense calcium fluxes across the epithelium as a whole. This investigation characterized the mechanisms of calcium transport by lobster hepatopancreatic ER vesicles and the effects of drugs and heavy metals on them. Kinetic constants for 45Ca2+ influx under control conditions were K(n) (m)=10.38+/-1.01 microM, J(max)=14.75+/-1.27 pmol/mg protein x sec, and n=2.53+/-0.46. The Hill coefficient for 45Ca2+ influx under control conditions, approximating 2, suggests that approximately two calcium ions were transported for each transport cycle in the absence of ATP or the inhibitors. Addition of 1 mM ATP to the incubation medium significantly (P<0.01) elevated the rate of 45Ca2+ influx at all calcium activities used and retained the sigmoidal nature of the transport relationship. The kinetic constants for 45Ca2+ influx in the presence of 1 mM ATP were K(n) (m)=12.76+/-0.91 microM, J(max)=25.46+/-1.45 pmol/mg protein x sec, and n=1.95+/-0.15. Kinetic analyses of ER 65Zn2+ influx resulted in a sigmoidal relationship between transport rate and zinc activity under control conditions (K(n) (m)=38.63+/-0.52 microM, J(max)=19.35+/-0.17 pmol/mg protein x sec, n=1.81+/-0.03). The Addition of 1 mM ATP enhanced 65Zn2+ influx at each zinc activity, but maintained the overall sigmoidal nature of the kinetic relationship. The kinetic constants for zinc influx in the presence of 1 mM ATP were K(n) (m)=34.59+/-2.31 microM, J(max)=26.09+/-1.17 pmol/mg protein x sec, and n=1.96+/-0.17. Both sigmoidal and ATP-dependent calcium and zinc influxes by ER vesicles were reduced in the presence of thapsigargin and vanadate. This investigation found that lobster hepatopancreatic ER exhibited a thapsigargin- and vanadate-inhibited, SERCA-like, calcium ATPase. This transporter displayed cooperative calcium transport kinetics (Hill coefficient, n approximately 2.0) and was inhibited by the heavy metals zinc and copper, suggesting that the metals may reduce the binding and transport of calcium when they are present in the cytosol.

Secretory pathway Ca(2+)-ATPase (SPCA1) Ca(2)+ pumps, not SERCAs, regulate complex [Ca(2+)](i) signals in human spermatozoa.

The sarcoplasmic-endoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitors thapsigargin (0.1-1 microM) and cyclopiazonic acid (10 microM), failed to affect resting [Ca(2+)] in human spermatozoa. Slow progesterone-induced [Ca(2+ i)](i) oscillations in human spermatozoa, which involve cyclic emptying-refilling of an intracellular Ca(2+) store were also insensitive to these inhibitors. Non-selective doses of thapsigargin (5-30 microM, 50-300 times the saturating dose for SERCA inhibition), caused elevation of resting [Ca(2+)](i) and partial, dose-dependent disruption of oscillations. A 10-40 microM concentration of bis(2-hydroxy-3-tert-butyl-5-methyl-phenyl)methane (bis-phenol), which inhibits both thapsigargin-sensitive and -insensitive microsomal Ca(2+) ATPases, caused elevation of resting [Ca(2+)](i) and inhibition of [Ca(2+)](i) oscillations at doses consistent with inhibition of thapsigargin-resistant, microsomal ATPase and liberation of stored Ca(2+). Low doses of bis-phenol had marked effects on [Ca(2+)](i) oscillation kinetics. Application of the drug to cells previously stimulated with progesterone had effects very similar to those observed when it was applied to unstimulated cells, suggesting that the sustained Ca(2+) influx induced by progesterone is not mediated via mobilisation of Ca(2+) stores. Western blotting for human sperm proteins showed expression of secretory pathway Ca(2+) ATPase (SPCA1). Immunolocalisation studies revealed expression of SPCA1 in all cells in an area behind the nucleus, extending into the midpiece. Staining for SERCA, carried out in parallel, detected no expression with either technique. We conclude that: (1) intracellular Ca(2+) store(s) and store-dependent [Ca(2+)](i) oscillations in human spermatozoa rely primarily on a thapsigargin/cyclopiazonic acid-insensitive Ca(2+) pump, which is not a SERCA as characterised in somatic cells; (2) effects of high-dose thapsigargin on spermatozoa primarily reflect non-specific actions on non-SERCAs and; (3) secretory pathway Ca(2+) ATPases contribute at least part of this non-SERCA Ca(2+) pump activity.