Endocrine disrupting alkylphenols: structural requirements for their adverse effects on Ca2+ pumps, Ca2+ homeostasis & Sertoli TM4 cell viability

Alkylphenols such as nonylphenol are pollutants that are widely dispersed within our environment. They bio-accumulate within man, with levels in the muM concentration range reported in human tissues. These chemicals act as endocrine disruptors, having xenoestrogenic activity. More recently alkylphenols have also been shown to affect Ca2+ signalling pathways. Here we show that alkylphenols are potent inhibitors of sarcoplasmic-endoplasmic reticulum Ca2+-ATPase (SERCA) activity. For linear chain alkylphenols the potency of inhibition is related to chain length, with the IC50 values for inhibition ranging from 8 microM for 4-n-nonylphenol (C9) to 1.3 mM for 4-n-propylphenol (C3). Branched chain alkylphenols generally had lower potencies than their linear chain counterparts, however, good correlations for all alkylphenols were observed between their Ca2+ pump inhibition and hydrophobicity, molecular volume and flexibility, indicating that these parameters are all important factors. Alkylphenols cause abnormal elevations of intracellular [Ca2+] within TM4 Sertoli cells (cells involved in sperm maturation) depolarise their mitochondria and induce cell death in these cells, in an alkyl chain size-dependent manner.     

Humic acid induced growth retardation in a sertoli cell line, TM4

Humic acid (HA) is a fluorescent deep brown organic, polymeric compound composed of phenolic acid. Intraperitoneal injection of HA in rats induced testicular morphological changes including degeneration of the seminiferous tubule, reduction in the number of Sertoli cells and spermatogonia, and a loss of spermatids. It was suggested that Sertoli cells may be involved in the progression of testicular atrophy. In this study, we used a mouse Sertoli cell Line, TM4, to investigate the effect of HA on Sertoli cells and the mechanism of the testicular atrophy induced by HA. We found that the cell growth of TM4 cells were reduced in 1 to 4 days of HA exposure. FACScan analysis of the DNA content of HA-treated TM4 cells revealed that there was no sub-G1 peak, indicating that the TM4 cells did not commit to the programmed cell death. However, a large proportion of TM4 cells were arrested at the G1 phase. The percentage of TM4 cells at the G1 phase increased from 36% to 84% after HA treatment for 4 days. Western blot assay of HA-treated TM4 cells showed that the expression of cyclin D1 protein decreased while the expression of p27kiP1 protein increased. These results suggest that HA-induced testicular atrophy is linked in part to an inhibitory effect on the growth of Sertoli cells. This model may be useful in investigation of environmental agents inducing testicular atrophy.     

Lack of evidence for presenilins as endoplasmic reticulum Ca2+ leak channels

Familial Alzheimer disease (FAD) is linked to mutations in the presenilin (PS) homologs. FAD mutant PS expression has several cellular consequences, including exaggerated intracellular Ca(2+) ([Ca(2+)](i)) signaling due to enhanced agonist sensitivity and increased magnitude of [Ca(2+)](i) signals. The mechanisms underlying these phenomena remain controversial. It has been proposed that PSs are constitutively active, passive endoplasmic reticulum (ER) Ca(2+) leak channels and that FAD PS mutations disrupt this function resulting in ER store overfilling that increases the driving force for release upon ER Ca(2+) release channel opening. To investigate this hypothesis, we employed multiple Ca(2+) imaging protocols and indicators to directly measure ER Ca(2+) dynamics in several cell systems. However, we did not observe consistent evidence that PSs act as ER Ca(2+) leak channels. Nevertheless, we confirmed observations made using indirect measurements employed in previous reports that proposed this hypothesis. Specifically, cells lacking PS or expressing a FAD-linked PS mutation displayed increased area under the ionomycin-induced [Ca(2+)](i) versus time curve (AI) compared with cells expressing WT PS. However, an ER-targeted Ca(2+) indicator revealed that this did not reflect overloaded ER stores. Monensin pretreatment selectively attenuated the AI in cells lacking PS or expressing a FAD PS allele. These findings contradict the hypothesis that PSs form ER Ca(2+) leak channels and highlight the need to use ER-targeted Ca(2+) indicators when studying ER Ca(2+) dynamics.     

Functional coupling between ryanodine receptors, mitochondria and Ca(2+) ATPases in rat submandibular acinar cells

Agonist stimulation of exocrine cells leads to the generation of intracellular Ca(2+) signals driven by inositol 1,4,5-trisphosphate receptors (IP(3)Rs) that rapidly become global due to propagation throughout the cell. In many types of excitable cells the intracellular Ca(2+) signal is propagated by a mechanism of Ca(2+)-induced Ca(2+) release (CICR), mediated by ryanodine receptors (RyRs). Expression of RyRs in salivary gland cells has been demonstrated immunocytochemically although their functional role is not clear. We used microfluorimetry to measure Ca(2+) signals in the cytoplasm, in the endoplasmic reticulum (ER) and in mitochondria. In permeabilized acinar cells caffeine induced a dose-dependent, transient decrease of Ca(2+) concentration in the endoplasmic reticulum ([Ca(2+)](ER)). This decrease was inhibited by ryanodine but was insensitive to heparin. Application of caffeine, however, did not elevate cytosolic Ca(2+) concentration ([Ca(2+)](i)) suggesting fast local buffering of Ca(2+) released through RyRs. Indeed, activation of RyRs produced a robust mitochondrial Ca(2+) transient that was prevented by addition of Ca(2+) chelator BAPTA but not EGTA. When mitochondrial Ca(2+) uptake was blocked, activation of RyRs evoked only a non-transient increase in [Ca(2+)](i) and substantially smaller Ca(2+) release from the ER. Upon simultaneous inhibition of mitochondrial Ca(2+) uptake and either plasmalemmal or ER Ca(2+) ATPase, activation of RyRs caused a transient rise in [Ca(2+)](i). Collectively, our data suggest that Ca(2+) released through RyRs is mostly "tunnelled" to mitochondria, while Ca(2+) ATPases are responsible for the fast initial sequestration of Ca(2+). Ca(2+) uptake by mitochondria is critical for maintaining continuous CICR. A complex interplay between RyRs, mitochondria and Ca(2+) ATPases is accomplished through strategic positioning of mitochondria close to both Ca(2+) release sites in the ER and Ca(2+) pumping sites of the plasmalemma and the ER.     

Sorting of calcium signals at the junctions of endoplasmic reticulum and mitochondria

Calcium signal transmission between endoplasmic reticulum (ER) and mitochondria is supported by a local [Ca(2+)] control that operates between IP(3)receptor Ca(2+)release channels (IP(3)R) and mitochondrial Ca(2+)uptake sites, and displays functional similarities to synaptic transmission. Activation of IP(3)R by IP(3)is known to evoke quantal Ca(2+)mobilization that is associated with incremental elevations of mitochondrial matrix [Ca(2+)] ([Ca(2+)](m)). Here we report that activation of IP(3)R by adenophostin-A (AP) yields non-quantal Ca(2+)mobilization in mast cells. We also show that the AP-induced continuous Ca(2+)release causes relatively small [Ca(2+)](m)responses, in particular, the sustained phase of Ca(2+)release is not sensed by the mitochondria. Inhibition of ER Ca(2+)pumps by thapsigargin slightly increases IP(3)-induced [Ca(2+)](m)responses, but augments AP-induced [Ca(2+)](m)responses in a large extent. In adherent permeabilized cells exposed to elevated [Ca(2+)], ER Ca(2+)uptake fails to affect global cytosolic [Ca(2+)], but attenuates [Ca(2+)](m)responses. Moreover, almost every mitochondrion exhibits a region very close to ER Ca(2+)pumps visualized by BODIPY-FL-thapsigargin or SERCA antibody. Thus, at the ER-mitochondrial junctions, localized ER Ca(2+)uptake provides a mechanism to attenuate the mitochondrial response during continuous Ca(2+)release through the IP(3)R or during gradual Ca(2+)influx to the junction between ER and mitochondria.     

The expression, activity and localisation of the secretory pathway Ca2+ -ATPase (SPCA1) in different mammalian tissues

The distribution of the secretory pathway Ca2+ -ATPase (SPCA1) was investigated at both the mRNA and protein level in a variety of tissues. The mRNA and the protein for SPCA1 were relatively abundant in rat brain, testis and testicular derived cells (myoid cells, germ cells, primary Sertoli cells and TM4 cells; a mouse Sertoli cell line) and epididymal fat pads. Lower levels were found in aorta (rat and porcine), heart, liver, lung and kidney. SPCA activities from a number of tissues were measured and shown to be particularly high in brain, aorta, heart, fat pads and testis. As the proportion of SPCA activity compared to total Ca2+ ATPase activity in brain, aorta, fat pads and testis were relatively high, this suggests that SPCA1 plays a major role in Ca2+ storage within these tissues. The subcellular localisation of SPCA1 was shown to be predominantly around the Golgi in both human aortic smooth muscle cells and TM4 cells.     

Effects of PMCA and SERCA pump overexpression on the kinetics of cell Ca(2+) signalling

The dynamic interactions of the main pathways for active Ca(2+) transport have been analysed in living cells by altering the expression of their components. The plasma membrane (PMCA) and the endoplasmic reticulum (ER) (SERCA) Ca(2+) pumps were transiently overexpressed in CHO cells, and the Ca(2+) homeostasis in the subcellular compartments was investigated using specifically targeted chimaeras of the Ca(2+)- sensitive photoprotein aequorin. In resting cells, overexpression of the PMCA and SERCA pumps caused a reduction and an increase in ER [Ca(2+)] levels, respectively, while no significant differences were detected in cytosolic and mitochondrial [Ca(2+)]. Upon stimulation with an inositol 1,4, 5-trisphosphate (IP(3))-generating agonist, the amplitude of the mitochondrial and cytosolic Ca(2+) rises correlated with the ER [Ca(2+)] only up to a threshold value, above which the feedback inhibition of the IP(3) channel by Ca(2+) appeared to be limiting.     

The endoplasmic reticulum as one continuous Ca(2+) pool: visualization of rapid Ca(2+) movements and equilibration

We investigated whether the endoplasmic reticulum (ER) is a functionally connected Ca(2+) store or is composed of separate subunits by monitoring movements of Ca(2+) and small fluorescent probes in the ER lumen of pancreatic acinar cells, using confocal microscopy, local bleaching and uncaging. We observed rapid movements and equilibration of Ca(2+) and the probes. The bulk of the ER at the base was not connected to the granules in the apical part, but diffusion into small apical ER extensions occurred. The connectivity of the ER Ca(2+) store was robust, since even supramaximal acetylcholine (ACh) stimulation for 30 min did not result in functional fragmentation. ACh could elicit a uniform decrease in the ER Ca(2+) concentration throughout the cell, but repetitive cytosolic Ca(2+) spikes, induced by a low ACh concentration, hardly reduced the ER Ca(2+) level. We conclude that the ER is a functionally continuous unit, which enables efficient Ca(2+) liberation. Ca(2+) released from the apical ER terminals is quickly replenished from the bulk of the rough ER at the base.     

Basal and physiological Ca(2+) leak from the endoplasmic reticulum of pancreatic acinar cells. Second messenger-activated channels and translocons

We have studied the Ca(2+) leak pathways in the endoplasmic reticulum of pancreatic acinar cells by directly measuring Ca(2+) in the endoplasmic reticulum ([Ca(2+)](ER)). Cytosolic Ca(2+) ([Ca(2+)](C)) was clamped to the resting level by a BAPTA-Ca(2+) mixture. Administration of cholecystokinin within the physiological concentration range caused a graded decrease of [Ca(2+)](ER), and the rate of Ca(2+) release generated by 10 pm cholecystokinin is at least 3x as fast as the basal Ca(2+) leak revealed by inhibition of the endoplasmic reticulum Ca(2+)-ATPase. Acetylcholine also evokes a dose-dependent decrease of [Ca(2+)](ER), with an EC(50) of 0.98 +/- 0.06 microm. Inhibition of receptors for inositol 1,4,5-trisphosphate (IP(3)) by heparin or flunarizine blocks the effect of acetylcholine but only partly blocks the effect of cholecystokinin. 8-NH(2) cyclic ADP-ribose (20 microm) inhibits the action of cholecystokinin, but not of acetylcholine(.) The basal Ca(2+) leak from the endoplasmic reticulum is not blocked by antagonists of the IP(3) receptor, the ryanodine receptor, or the receptor for nicotinic acid adenine dinucleotide phosphate. However, treatment with puromycin (0.1-1 mm) to remove nascent polypeptides from ribosomes increases Ca(2+) leak from the endoplasmic reticulum by a mechanism independent of the endoplasmic reticulum Ca(2+) pumps and of the receptors for IP(3) or ryanodine.     

Regulation of tissue inhibitor of metalloproteinases-1 in rat Sertoli cells: induction by germ cell residual bodies, interleukin-1alpha, and second messengers

In the testis, FSH has been shown to induce the expression and secretion of tissue inhibitor of metalloproteinases-1 (TIMP-1) from Sertoli cells in vitro. This study was performed to elucidate further the cellular origin of testicular TIMP-1 and its expression by hormonal and paracrine factors. This is the first report on the expression of testicular TIMP-1 in vivo. TIMP-1 mRNA in whole testis was decreased after hypophysectomy and strongly increased by the injection of FSH-S17 to hypophysectomized rats. Primary cultures of both peritubular and Sertoli cells showed basal expression of TIMP-1 mRNA. In contrast, we were unable to detect TIMP-1 mRNA in Leydig cells, freshly isolated immature germ cells (primary spermatocytes and spermatids), or residual bodies. We further show that treatment of Sertoli cells with 8-(4-chlorophenyl)thio-cAMP (8-CPTcAMP) in combination with 12-O-tetradecanoylphorbol 13-acetate (TPA) or Ca(2+) inducers (calcium ionophore A23187 or thapsigargin) had additive (TPA) and synergistic effects (Ca(2+)) on the level of TIMP-1 mRNA and secreted protein. We also show that both the level of TIMP-1 mRNA and secreted protein from Sertoli cells were strongly increased by residual bodies, as well as by the cytokine interleukin-1alpha. TIMP-1 was not up-regulated by either 8-CPTcAMP or interleukin-1alpha in peritubular cells. In contrast to the regulated secretory fraction of TIMP-1, we also detected constitutively expressed immunoreactive TIMP-1 in the nucleus of Sertoli cells, suggesting a role of nuclear TIMP-1 in these cells. In conclusion, our data show that secretion of TIMP-1 from Sertoli cells is highly regulated by hormonal and local processes in the testis, indicating that TIMP-1 is of physiological importance during both testicular development and spermatogenesis.     

Herp stabilizes neuronal Ca2+ homeostasis and mitochondrial function during endoplasmic reticulum stress

In response to endoplasmic reticulum (ER) stress, cells launch homeostatic and protective responses, but can also activate cell death cascades. A 54 kDa integral ER membrane protein called Herp was identified as a stress-responsive protein in non-neuronal cells. We report that Herp is present in neurons in the developing and adult brain, and that it is regulated in neurons by ER stress; sublethal levels of ER stress increase Herp levels, whereas higher doses decrease Herp levels and induce apoptosis. The decrease in Herp protein levels following a lethal ER stress occurs prior to mitochondrial dysfunction and cell death, and is mediated by caspases which generate a 30-kDa proteolytic Herp fragment. Mutagenesis of the caspase cleavage site in Herp enhances its neuroprotective function during ER stress. While suppression of Herp induction by RNA interference sensitizes neural cells to apoptosis induced by ER stress, overexpression of Herp promotes survival by a mechanism involving stabilization of ER Ca(2+) levels, preservation of mitochondrial function and suppression of caspase 3 activation. ER stress-induced activation of JNK/c-Jun and caspase 12 are reduced by Herp, whereas induction of major ER chaperones is unaffected. Herp prevents ER Ca(2+) overload under conditions of ER stress and agonist-induced ER Ca(2+) release is attenuated by Herp suggesting a role for Herp in regulating neuronal Ca(2+) signaling. By stabilizing ER Ca(2+) homeostasis and mitochondrial functions, Herp serves a neuroprotective function under conditions of ER stress.     

Fas ligand expression in TM4 Sertoli cells is enhanced by estradiol "in situ" production

The testis is an immunologically privileged site of the body where Sertoli cells work on to favor local immune tolerance by testicular autoantigens segregation and immunosuppressive factors secretion. Fas/Fas Ligand (FasL) system, expressed prevalently in Sertoli cells, has been considered to be one of the central mechanisms in testis immunological homeostasis. In different cell lines it has been reported that the proapoptotic protein FasL is regulated by 17-beta estradiol (E2). Thus, using as experimental model mouse Sertoli cells TM4, which conserve a large spectrum of functional features present in native Sertoli cells, like aromatase activity, we investigated if estradiol "in situ" production may influence FasL expression. Our results demonstrate that an aromatizable androgen like androst-4-ene-3,17-dione (Delta4) enhanced FasL mRNA, protein content and promoter activity in TM4 cells. The treatment with N(6),2'-O-dibutyryladenosine-3'-5'-cyclic monophosphate [(Bu)(2)cAMP] (simulating FSH action), that is well known to stimulate aromatase activity in Sertoli cells, amplified Delta4 induced FasL expression. Functional studies of mutagenesis, electrophoretic mobility shift (EMSA) and chromatin immunoprecipitation (ChIP) assays revealed that the Sp-1 motif on FasL promoter was required for E2 enhanced FasL expression in TM4 cells. These data let us to recruit FasL among those genes whose expression is up-regulated by E2 through a direct interaction of ERalpha with Sp-1 protein. Finally, evidence that an aromatizable androgen is able to increase FasL expression suggests that E2 production by aromatase activity may contribute to maintain the immunoprivilege status of Sertoli cells.     

The ATP-binding cassette transporter 1 mediates lipid efflux from Sertoli cells and influences male fertility

The liver X receptor/retinoid X receptor (LXR/RXR)-regulated gene ABCA1 effluxes cellular cholesterol and phospholipid to apolipoprotein A1 (apoA1), which is the rate-limiting step in high-density lipoprotein synthesis. The RXR pathway plays a critical role in testicular lipid trafficking, and RXRbeta-deficient male mice are sterile and accumulate lipids in Sertoli cells. Here, we demonstrate that ABCA1 mRNA and protein are abundant in Sertoli cells, whereas germ cells express little ABCA1. LXR/RXR agonists stimulate ABCA1 expression in cultured Sertoli MSC1 and Leydig TM3 cell lines. However, Sertoli TM4 cells lack ABCA1, and TM4 cells or primary Sertoli cells cultured from ABCA1(-/-) mice both fail to efflux cholesterol to apoA1. Expression of exogenous ABCA1 restores apoA1-dependent cholesterol efflux in Sertoli TM4 cells. In vivo, ABCA1-deficient mice exhibit lipid accumulation in Sertoli cells and depletion of normal lipid droplets from Leydig cells by 2 months of age. By 6 months of age, intratesticular testosterone levels and sperm counts are significantly reduced in ABCA1(-/-) mice compared with wild-type (WT) controls. Finally, a 21% decrease (P = 0.01) in fertility was observed between ABCA1(-/-) males compared with WT controls across their reproductive lifespans. These results show that ABCA1 plays an important role in lipid transport in Sertoli cells and influences male fertility.     

PERK activation at low glucose concentration is mediated by SERCA pump inhibition and confers preemptive cytoprotection to pancreatic β-cells

Protein kinase R-like ER kinase (PERK) is activated at physiologically low glucose concentrations in pancreatic β-cells. However, the molecular mechanisms by which PERK is activated under these conditions and its role in β-cell function are poorly understood. In this report, we investigated, in dispersed rat islets of Langerhans and mouse insulinoma-6 (MIN6) cells, the relationship between extracellular glucose concentration, the free endoplasmic reticulum (ER) calcium concentration ([Ca(2+)](ER)) measured directly using an ER targeted fluorescence resonance energy transfer-based calcium sensor, and the activation of PERK. We found that a decrease in glucose concentration leads to a concentration-dependent reduction in [Ca(2+)](ER) that parallels the activation of PERK and the phosphorylation of its substrate eukaryotic initiation factor-2α. We provide evidence that this decrease in [Ca(2+)](ER) is caused by a decrease in sarcoplasmic/ER Ca(2+)-ATPase pump activity mediated by a reduction in the energy status of the cell. Importantly, we also report that PERK-dependent eukaryotic initiation factor-2α phosphorylation at low glucose concentration plays a significant role in 1) the regulation of both proinsulin and global protein synthesis, 2) cell viability, and 3) conferring preemptive cytoprotection against ER stress. Taken together, these results provide evidence that a decrease in the ATP/energy status of the cell in response to a decrease in glucose concentration results in sarcoplasmic/ER Ca(2+)-ATPase pump inhibition, the efflux of Ca(2+) from the ER, and the activation of PERK, which plays an important role in both pancreatic β-cell function and survival.     

Visualization and quantification of endoplasmic reticulum Ca2+ in renal cells using confocal microscopy and Fluo5F

Sarcoplasmic/endoplasmic reticulum (ER) Ca(2+) is the most abundant store of intracellular Ca(2+), and its release is an important trigger of physiological and cell death pathways. Previous work in our laboratory revealed the importance of ER Ca(2+) in toxicant-induced renal proximal tubular cell (RPTC) death. The purpose of this study was to evaluate the use of confocal microscopy and Fluo5F, a low affinity Ca(2+) indicator, to directly monitor changes in RPTC ER Ca(2+). Fluo5F staining reflected ER Ca(2+), resolved ER structure, and showed no colocalization with tetramethyl rhodamine methyl ester (TMRM), a marker of mitochondrial membrane potential. Thapsigargin, an ER Ca(2+) pump inhibitor, decreased ER fluorescence by 30% and 55% at 5 and 15 min, respectively, whereas A23187, a Ca(2+) ionophore caused more rapid ER Ca(2+) release (55% and 75% decrease in fluorescence at 5 and 15 min). Carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), a mitochondrial uncoupler, added at the end of the experiment, further decreased ER fluorescence after thapsigargin treatment, revealing that thapsigargin did not release all ER Ca(2+). In contrast, FCCP did not decrease ER fluorescence after A23187 treatment, suggesting complete ER Ca(2+) release. ER Ca(2+) release in response to A23187 or thapsigargin resulted in a modest but significant decrease in mitochondrial membrane potential. These data provide evidence that confocal microscopy and Fluo5F are useful and effective tools for directly monitoring ER Ca(2+) in live cells.     

Contraceptive steroids from pharmaceutical waste perturbate junctional communication in Sertoli cells

The potential health impact of pharmaceutical waste is now a growing concern. Contraceptive steroids are prominent environmental contaminants and thus may act as endocrine disruptors. Numerous xenobiotics hamper Sertoli cells junctional communication which is known to participate in spermatogenesis control. This has been associated with male subfertility and testicular cancer. We investigated three contraceptive molecules found in the environment for their potential impact on Sertoli cells gap junction functionality: 17a-ethynylestradiol, medroxyprogesterone acetate and levonorgestrel. Four other non-steroid drugs also found in the environment were included in the study. Communication disruption was analyzed in vitro in murine seminiferous tubules and the 42GPA9 Sertoli cell line. Steroids modulated connexin43 trafficking and impaired junctional communication through rapid effects apparently acting on the cell membrane but not on Cx43 expression. The 4 non-steroid compounds showed no effect. Longer exposure to steroids increased gap junction impairment, which was associated in part with Na/K ATPase internalization. Estrogen receptors (ER) did not appear to be involved in gap junction disruption: Sertoli cells are devoid of ERα and only express the cytoplasmic β isoform. ERβ localization was not modified by either steroid. The threshold level was surprisingly low, around 10^?16 M. We conclude that steroidal pollutants disrupt Sertoli cells junctional communication in vitro at concentrations that can be found in the environment.     

Single amino acid mutations in transmembrane domain 5 confer to the plasma membrane Ca2+ pump properties typical of the Ca2+ pump of endo(sarco)plasmic reticulum

Conserved residues in some of the transmembrane domains are proposed to mediate ion translocation by P-type pumps. The plasma membrane Ca(2+) pump (PMCA) lacks 2 of these residues in transmembrane domains (TM) 5 and 8. In particular, a glutamic acid (Glu-771) residue in TM5, which is proposed to be involved in the binding and transport of Ca(2+) by the sarcoplasmic reticulum Ca(2+) pump (SERCA), is replaced by an alanine (Ala-854) in the PMCA pump. Ala-854 has been mutated to Glu, Asp, or Gln; Glu-975 in TM8, which is an Ala in the SERCA pump, has been mutated to Gln, Asp, or Ala. The mutants have been expressed in three cell systems, with or without the help of viruses. When expressed in large amounts in Sf9 cells, the mutated pumps were isolated and analyzed in the purified state. Two of the three TM8 mutants were correctly delivered to the plasma membrane and were active. All the TM5 mutants were retained in the endoplasmic reticulum; two of them (A854Q and A854E) retained activity. Their properties (La(3+) sensitivity and decay of the phosphorylated intermediate, higher cooperativity of Ca(2+) binding with a Hill's coefficient approaching 2) differed from those of the expressed wild type PMCA pump, and resembled those of the SERCA pump.     

Steady-state free Ca(2+) in the yeast endoplasmic reticulum reaches only 10 microM and is mainly controlled by the secretory pathway pump pmr1.

Over recent decades, diverse intracellular organelles have been recognized as key determinants of Ca(2+) signaling in eukaryotes. In yeast however, information on intra-organellar Ca(2+) concentrations is scarce, despite the demonstrated importance of Ca(2+) signals for this microorganism. Here, we directly monitored free Ca(2+) in the lumen of the endoplasmic reticulum (ER) of yeast cells, using a specifically targeted version of the Ca(2+)-sensitive photoprotein aequorin. Ca(2+) uptake into the yeast ER displayed characteristics distinctly different from the mammalian ER. At steady-state, the free Ca(2+) concentration in the ER lumen was limited to approximately 10 microM, and ER Ca(2+) sequestration was insensitive to thapsigargin, an inhibitor specific for mammalian ER Ca(2+) pumps. In pmr1 null mutants, free Ca(2+) in the ER was reduced by 50%. Our findings identify the secretory pathway pump Pmr1, predominantly localized in the Golgi, as a major component of ER Ca(2+) uptake activity in yeast.     

Acid phosphatases in germinal and somatic cells of the testes

Four forms of acid phosphatase have been found in the testicular tissue of many mammalian species, but their exact cellular site has remained obscure. In this work, acid phosphatases have been studied in different reproductive organs of the male rat, in somatic cell lines derived by cloning from both rat and mouse testes, in primary cultures of rat Sertoli cells, and in isolated spermatogenic cells of the mouse. Among the reproductive organs, preputial glands show the highest specific activities with p-nitrophenyl phosphate as substrate, followed by the testicular tissue and the different regions of the epididymis. By contrast to that in other tissues, testicular activity with p-nitrophenyl phosphate is not influenced by tartrate and is activated markedly by cobalt (Co2+). Among the somatic cell lines, the highest hydrolysis rates are obtained with naphthyl substrates in the epithelial (TR-1) and myoid (TR-M) cell lines and marginally lower rates in the Leydig (TM3) and Sertoli (TM4) cell lines. With thymolphthalein phosphate, the latter two cell lines show very low activity. These activities are not influenced by different hormones and growth factors in the culture medium. The most marked Co2+-activated reaction with p-nitrophenyl phosphate is found in advanced stages of germinal cells and residual bodies. Primary cultures of Sertoli cells, prepared from rats 10 to 30 days of age, show a slight decrease in acid phosphatase levels; however, the activities are not influenced markedly by addition of follicle-stimulating hormone (FSH) and/or testosterone to the culture medium.(ABSTRACT TRUNCATED AT 250 WORDS)