Soluble adenylyl cyclase regulates the cytosolic NADH/NAD+ redox state and the bioenergetic switch between glycolysis and oxidative phosphorylation

The evolutionarily conserved soluble adenylyl cyclase (sAC, ADCY10) mediates cAMP signaling exclusively in intracellular compartments. Because sAC activity is sensitive to local concentrations of ATP, bicarbonate, and free Ca²⁺, sAC is potentially an important metabolic sensor. Nonetheless, little is known about how sAC regulates energy metabolism in intact cells. In this study, we demonstrated that both pharmacological and genetic suppression of sAC resulted in increased lactate secretion and decreased pyruvate secretion in multiple cell lines and primary cultures of mouse hepatocytes and cholangiocytes. The increased extracellular lactate-to-pyruvate ratio upon sAC suppression reflected an increased cytosolic free [NADH]/[NAD⁺] ratio, which was corroborated by using the NADH/NAD⁺ redox biosensor Peredox-mCherry. Mechanistic studies in permeabilized HepG2 cells showed that sAC inhibition specifically suppressed complex I of the mitochondrial respiratory chain. A survey of cAMP effectors revealed that only selective inhibition of exchange protein activated by cAMP 1 (Epac1), but not protein kinase A (PKA) or Epac2, suppressed complex I-dependent respiration and significantly increased the cytosolic NADH/NAD⁺ redox state. Analysis of the ATP production rate and the adenylate energy charge showed that inhibiting sAC reciprocally affects ATP production by glycolysis and oxidative phosphorylation while maintaining cellular energy homeostasis. In conclusion, our study shows that, via the regulation of complex I-dependent mitochondrial respiration, sAC-Epac1 signaling regulates the cytosolic NADH/NAD⁺ redox state, and coordinates oxidative phosphorylation and glycolysis to maintain cellular energy homeostasis. As such, sAC is effectively a bioenergetic switch between aerobic glycolysis and oxidative phosphorylation at the post-translational level.     

Effect of the absence of bicarbonate upon intracellular pH and calcium fluxes in pancreatic islet cells

The removal of extracellular HCO⁻₃ together with a decrease in pCO₂, in order to maintain a normal extracellular pH, caused a sustained increase of intracellular pH in rat pancreatic islets. This increase was more marked in glucose-deprived than in glucose-stimulated islets, and was associated with a facilitation of ⁴⁵Ca efflux from the glucose-deprived islets. Such a facilitation was slightly reduced in the absence of extracellular Ca²⁺ and abolished at low extracellular Na⁺ concentration. It failed to occur in glucose-stimulated islets, whether in the presence or absence of extracellular Ca²⁺. The removal of HCO⁻₃ and decrease in the pCO₂ also reduced the magnitude of both the secondary rise in ⁴⁵Ca efflux and stimulation of insulin release normally evoked by an increase in glucose concentration. These findings suggest that changes in intracellular pH affect both the outflow of Ca²⁺ from islet cells as mediated by Na⁺-Ca²⁺ countertransport and the inflow of Ca²⁺ by gated Ca²⁺ channels. The experimental data are also compatible with the view that islet cells are equipped with an active process of bicarbonate-chloride exchange involved in the regulation of intracellular pH.     

Role of N-glycosylation in cell surface expression and protection against proteolysis of the intestinal anion exchanger SLC26A3

SLC26A3 is a Cl(-)/HCO(3)(-) exchanger that plays a major role in Cl(-) absorption from the intestine. Its mutation causes congenital chloride-losing diarrhea. It has been shown that SLC26A3 are glycosylated, with the attached carbohydrate being extracellular and perhaps modulating function. However, the role of glycosylation has yet to be clearly determined. We used the approaches of biochemical modification and site-directed mutagenesis to prevent glycosylation. Deglycosylation experiments with glycosidases indicated that the mature glycosylated form of SLC26A3 exists at the plasma membrane, and a putative large second extracellular loop contains all of the N-linked carbohydrates. Deglycosylation of SLC26A3 causes depression of transport activity compared with wild-type, although robust intracellular pH changes were still observed, suggesting that N-glycosylation is not absolutely necessary for transport activity. To localize glycosylation sites, we mutated the five consensus sites by replacing asparagine (N) with glutamine. Immnoblotting suggests that SLC26A3 is glycosylated at N153, N161, and N165. Deglycosylation of SLC26A3 causes a defect in cell surface processing with decreased cell surface expression. We also assessed whether SLC26A3 is protected from tryptic digestion. While the mature glycosylated SLC26A3 showed little breakdown after treatment with trypsin, deglycosylated SLC26A3 exhibited increased susceptibility to trypsin, suggesting that the oligosaccharides protect SLC26A3 from tryptic digestion. In conclusion, our data indicate that N-glycosylation of SLC26A3 is important for cell surface expression and for protection from proteolytic degradation that may contribute to the understanding of pathogenesis of congenital disorders of glycosylation.     

Regulation of Epithelial Na+ Transport by Soluble Adenylyl Cyclase in Kidney Collecting Duct Cells

Alkalosis impairs the natriuretic response to diuretics, but the underlying mechanisms are unclear. The soluble adenylyl cyclase (sAC) is a chemosensor that mediates bicarbonate-dependent elevation of cAMP in intracellular microdomains. We hypothesized that sAC may be an important regulator of Na⁺ transport in the kidney. Confocal images of rat kidney revealed specific immunolocalization of sAC in collecting duct cells, and immunoblots confirmed sAC expression in mouse cortical collecting duct (mpkCCD_c14) cells. These cells exhibit aldosterone-stimulated transepithelial Na⁺ currents that depend on both the apical epithelial Na⁺ channel (ENaC) and basolateral Na⁺,K⁺-ATPase. RNA interference-mediated 60-70% knockdown of sAC expression comparably inhibited basal transepithelial short circuit currents (I_sc) in mpkCCD_c14 cells. Moreover, the sAC inhibitors KH7 and 2-hydroxyestradiol reduced I_sc in these cells by 50-60% within 30 min. 8-Bromoadenosine-3′,5′-cyclic-monophosphate substantially rescued the KH7 inhibition of transepithelial Na⁺ current. Aldosterone doubled ENaC-dependent I_sc over 4 h, an effect that was abolished in the presence of KH7. The sAC contribution to I_sc was unaffected with apical membrane nystatin-mediated permeabilization, whereas the sAC-dependent Na⁺ current was fully inhibited by basolateral ouabain treatment, suggesting that the Na⁺,K⁺-ATPase, rather than ENaC, is the relevant transporter target of sAC. Indeed, neither overexpression of sAC nor treatment with KH7 modulated ENaC currents in Xenopus oocytes. ATPase and biotinylation assays in mpkCCD_c14 cells demonstrated that sAC inhibition decreases catalytic activity rather than surface expression of the Na⁺,K⁺-ATPase. In summary, these results suggest that sAC regulates both basal and agonist-stimulated Na⁺ reabsorption in the kidney collecting duct, acting to enhance Na⁺,K⁺-ATPase activity.Maintenance of intracellular pH depends in part on the extracellular to intracellular Na⁺ gradient, and elevation of intracellular [Na⁺] can lead to acidification of the cytoplasm. It has been shown that acidification of the cytoplasm of cells from frog skin and toad bladder by increased partial pressure of CO₂ reduces Na⁺ transport and permeability (1, 2). Conversely, the rise in plasma bicarbonate caused by metabolic alkalosis with chronic diuretic use has been shown to increase net renal Na⁺ reabsorption independently of volume status, electrolyte depletion, and/or increased aldosterone secretion (3, 4). However, the underlying mechanisms involved in these phenomena remain unclear.The soluble adenylyl cyclase (sAC)² is a chemosensor that mediates the elevation of cAMP in intracellular microdomains (5-7). Unlike transmembrane adenylyl cyclases (tmACs), sAC is insensitive to regulation by forskolin or heterotrimeric G proteins (8) and is directly activated by elevations of intracellular calcium (9, 10) and/or bicarbonate ions (11). Thus, sAC mediates localized intracellular increases in cAMP in response to variations in bicarbonate levels or its closely related parameters, partial pressure of CO₂ and pH. Mammalian sAC is more similar to bicarbonate-regulated cyanobacterial adenylyl cyclases than to other mammalian nucleotidyl cyclases, which may indicate that there is a unifying mechanism for the regulation of cAMP signaling by bicarbonate across biological systems. Although sAC appears to be encoded by a single gene, there is significant isoform diversity for this ubiquitously expressed enzyme (11, 12) generated by alternative splicing (reviewed in Ref. 13). sAC has been shown to regulate the subcellular localization and/or activity of membrane transport proteins such as the vacuolar H⁺-ATPase (V-ATPase) and cystic fibrosis transmembrane conductance regulator in epithelial cells (14, 15). Functional activity of sAC has been reported in the kidney (16), and sAC has been localized to epithelial cells in the distal nephron (14, 17).Given that natriuresis is decreased during metabolic alkalosis, when bicarbonate is elevated, and Na⁺ reabsorption is impaired by high partial pressure of CO₂, we hypothesized that bicarbonate-regulated sAC may play a key role in the regulation of transepithelial Na⁺ transport in the distal nephron. Reabsorption of Na⁺ in the kidney and other epithelial tissues is mediated by the parallel operation of apical ENaC and basolateral Na⁺,K⁺-ATPase, and both transport proteins can be stimulated by cAMP via the cAMP-dependent protein kinase (PKA) (18, 53). The aims of this study were to investigate the role of sAC in the regulation of transepithelial Na⁺ transport in the kidney through the use of specific sAC inhibitors and electrophysiological measurements. We found that sAC inhibition blocks transepithelial Na⁺ reabsorption in polarized mpkCCD_c14 cells under both basal and hormone-stimulated conditions. Selective membrane permeabilization studies revealed that although ENaC activity appears to be unaffected by sAC inhibition, flux through the Na⁺,K⁺-ATPase is sensitive to sAC modulation. Inhibiting sAC decreases ATPase activity without affecting plasma membrane expression of the pump; thus, tonic sAC activity appears to be required for Na⁺ reabsorption in kidney collecting duct.     

Biosynthesis and intracellular movement of the melanosomal membrane glycoprotein gp75, the human b (brown) locus product

A 75-kDa melanosomal glycoprotein (gp75) is the product of a gene that maps to the b (brown) locus, a genetic locus that determines coat color in the mouse. The b locus is conserved (88% identity) between mouse and human. The mouse monoclonal antibody TA99 was used to study the biosynthesis and processing of gp75. gp75 was synthesized as a 55-kDa polypeptide, glycosylated by addition and processing of five or more Asnlinked carbohydrate chains through the cis and trans Golgi, and transported to melanosomes as a mature 75kDa form. Synthesis and processing of gp75 was rapid (T_1/2 < 30 min), and early steps in processing were required for efficient export of gp75 to melanosomes. Fully processed mature gp75 was quite stable (T_1/2 = 22–24 h) in the melanosome. Digestion of high-mannose carbohydrate chains with endo-β-N-acetylglucosaminidase H revealed two alternative processed forms of gp75 that differed in the number or composition of complex-type carbohydrate chains. The rate of synthesis and movement through intracellular membrane compartments was the same for both glycosylated forms. Studies with inhibitors of steps in oligosaccharide processing showed that alternative forms of gp75 were generated during trimming reactions by mannosidase IA/IB and that further maturation resulted in the two mature forms of gp75. We propose that the kinetics of biosynthesis and processing reflect events in the biogenesis and maturation of melanosomes.     

Cortical Granule Translocation during Maturation of Starfish Oocytes Requires Cytoskeletal Rearrangement Triggered by InsP3-Mediated CaRelease

Cortical granules (secretory vesicles located under the cortex of mature oocytes) release their contents to the medium at fertilization. Their exocytosis modifies the extracellular environment, blocking the penetration of additional sperm. The granules translocate to the surface during the maturation process, and it has been suggested that they move to the cortex via cytoskeletal elements. In this paper we show that the increase in intracellular Ca²⁺, which the maturing hormone 1-methyladenine (1-MA) induces in starfish through the activation of inositol 1,4,5-trisphosphate (InsP₃) receptors, triggers changes in filamentous actin, which then direct the correct movement and reorientation of the cortical granules and the elevation of the fertilization envelope.     

Inhibition of reticulocyte iron uptake by NH4Cl and CH3NH2

The aim of this investigation was to test the hypothesis that elevation of intracellular pH would inhibit iron uptake by reticulocytes. The experiments were performed with rabbit reticulocytes and iron bound to rabbit transferrin. Incubation of the cells with NH₄Cl, (NH₄)₂CO₃, CH₃NH₂ and (CH₃)₂NH was used in an attempt to increase intracellular pH. These substances were all found to inhibit iron uptake by reticulocytes. The mechanism of action of NH₄Cl and CH₃NH₂ was investigated in detail. Similar results were found with both reagents. They inhibited iron uptake in a concentration-dependent manner, but produced a small increase in the cellular uptake of transferrin. The onset of action was rapid and the effect was reversible. There was no decrease in the number of transferrin-binding sites per cell and their apparent affinity for transferrin increased slightly, while the efficiency of iron removal from transferrin per binding site diminished greatly. The rate of transferrin release from reticulocytes was unaffected. NH₄Cl did not affect the rate of iron release from transferrin in a cell-free system. Incubation of reticulocytes with 10 mM NH₄Cl or CH₃NH₂ was found to produce an increase in intracellular pH of 0.05—0.15 pH units. The intracellular pH determined by used of the weak acid 5,5-dimethyl-oxazolidine-2,4-dione was significantly higher than that obtained with the weak base (CH₃)₂NH. By transmission electron microscopy it was shown that reticulocytes treated with NH₄Cl or CH₃NH₂ have enlarged intracellular vesicles. The results are considered to support the hypothesis that iron release from transferrin in reticulocytes occurs as a result of protonation of the transferrin within intracellular vesicles. According to this hypothesis, weak bases such as NH₃ and CH₃NH₂ inhibit iron release by neutralizing H⁺ within the vesicles.     

A Cytotoxic,Co-operative Interaction Between Energy Deprivation and Glutamate Release From System xc? Mediates Aglycemic Neuronal Cell Death

The astrocyte cystine/glutamate antiporter (system x_c⁻) contributes substantially to the excitotoxic neuronal cell death facilitated by glucose deprivation. The purpose of this study was to determine the mechanism by which this occurred. Using pure astrocyte cultures, as well as, mixed cortical cell cultures containing both neurons and astrocytes, we found that neither an enhancement in system x_c⁻ expression nor activity underlies the excitotoxic effects of aglycemia. In addition, using three separate bioassays, we demonstrate no change in the ability of glucose-deprived astrocytes—either cultured alone or with neurons—to remove glutamate from the extracellular space. Instead, we demonstrate that glucose-deprived cultures are 2 to 3 times more sensitive to the killing effects of glutamate or N-methyl-D-aspartate when compared with their glucose-containing controls. Hence, our results are consistent with the weak excitotoxic hypothesis such that a bioenergetic deficiency, which is measureable in our mixed but not astrocyte cultures, allows normally innocuous concentrations of glutamate to become excitotoxic. Adding to the burgeoning literature detailing the contribution of astrocytes to neuronal injury, we conclude that under our experimental paradigm, a cytotoxic, co-operative interaction between energy deprivation and glutamate release from astrocyte system x_c⁻ mediates aglycemic neuronal cell death.     

Downregulation and subcellular redistribution of the γ-aminobutyric acidA receptor induced by tunicamycin in cultured brain neurons

The significance of N-linked glycosylation and oligosaccharide processing was examined for the expression of γ-aminobutyric acid_A receptor (GABA_AR) in cultured neurons derived from chick embryo brains. Incubation of cultures with 5 μg/ml of tunicamycin for 24 h blocked the binding of ³H-flunitrazepam and ³H-muscimol, probes for the benzodiazepine and GABA sites on the receptor, by about 20% and 28%, respectively. The loss of ligand binding was due to a reduction in the number of binding sites with no significant changes in receptor affinity. Light microscopic immunocytochemistry also revealed that the treatment reduced approximately 13% of the intensity of GABA_AR immunoreactivity in the neuronal somata. Furthermore, the fraction of intracellular receptors was decreased to 24% from 34% of control in the presence of the agent, as revealed by trypsinization of cells in situ followed by ³H-flunitrazepam binding. The molecular weight of the receptor subunit protein was lowered around 0.5 kDa after tunicamycin treatment, in accordance with that following N-glycosidase F digestion, indicating the blockade of N-linked glycosylation of GABA_AR by tunicamycin. Moreover, intense inhibitions of 91% and 44%, respectively, were detected to the general galactosylation and mannosylation in the tunicamycin-treated cells, whereas the protein synthesis was hindered by 13%, through assaying the incorporation of ³H-sugars and ³H-leucine. Nevertheless, treatment with castanospermine or swainsonine (10 μg/ml, 24 h), inhibitors to maturation of oligosaccharides, failed to produce significant changes in the ligand binding. In addition, in situ hybridization analysis showed that these three inhibitors did not perturb the mRNA of GABA_AR α₁-subunit. The data suggest that tunicamycin causes the downregulation and subcellular redistribution of GABA_AR by producing irregularly glycosylated receptors and modifying their localization. Both galactosylation and mannosylation during the process of N-linked glycosylation may be important for the functional expression and intracellular transport of GABA_AR. J. Cell. Biochem. 70:38–48, 1998. © 1998 Wiley-Liss, Inc.     

Extracellular Caper se inhibits quantal size of catecholamine release in adrenal slice chromaffin cells

Classic calcium hypothesis states that depolarization-induced increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) triggers vesicle exocytosis by increasing vesicle release probability in neurons and neuroendocrine cells. The extracellular Ca²⁺, in this calcium hypothesis, serves as a reservoir of Ca²⁺ source. Recently we find that extracellular Ca²⁺per se inhibits the [Ca²⁺]_i dependent vesicle exocytosis, but it remains unclear whether quantal size is regulated by extracellular, or intracellular Ca²⁺ or both [1]. In this work we showed that, in physiological condition, extracellular Ca²⁺per se specifically inhibited the quantal size of single vesicle release in rat adrenal slice chromaffin cells. The extracellular Ca²⁺ in physiological concentration (2.5 mM) directly regulated fusion pore kinetics of spontaneous quantal release of catecholamine. In addition, removal of extracellular Ca²⁺ directly triggered vesicle exocytosis without eliciting intracellular Ca²⁺. We propose that intracellular Ca²⁺ and extracellular Ca²⁺per se cooperately regulate single vesicle exocytosis. The vesicle release probability was jointly modulated by both intracellular and extracellular Ca²⁺, while the vesicle quantal size was mainly determined by extracellular Ca²⁺ in chromaffin cells physiologically.     

Chitinases and beta-1,3-Glucanases in the Apoplastic Compartment of Oat Leaves (Avena sativa L.)

To isolate chitinases and β-1,3-glucanases from the intercellular space of oats (Avena sativa L.), primary leaves were infiltrated with buffer and subjected to gentle centrifugation to obtain intercellular washing fluid (IWF). Approximately 5% of the chitinase and 10% of the β-1,3-glucanase activity of the whole leaf were released. Only small amounts (0.01-0.03%) of the intracellular marker malate-dehydrogenase were released into the IWF during infiltration. Activities of chitinase and β-1,3-glucanase in the IWF and in the leaf extract were compared by different chromatographic methods. On Sephadex G-75, chitinase appeared as a single peak (M_r 29.8 kD) both in IWF and homogenate. β-1,3-Glucanase, however, showed two peaks in the IWF (M_r 52 and 31.3 kD), whereas the elution pattern of the homogenate showed only one major peak at 22 kD. Chromatofocusing indicated that the IWF contained four chitinases and five β-1,3-glucanases. The elution pattern of the homogenate and IWF were similar with regard to the elution pH, but the peak intensities were distinctly different. Our results demonstrate that extracellular β-1,3-glucanases are different from those located intracellularly. Extracellular and intracellular chitinases do not differ in molecular properties, except for one isozyme which seems to be confined to the extracellular space. We suggest that both enzymes might play a special role in pathogenesis during fungal infection.     

Inositol 1,4,5-trisphosphate-induced calcium release from canine aortic sarcoplasmic reticulum vesicles

Inositol 1,4,5-trisphosphate-induced calcium release from canine aortic smooth muscle sarcoplasmic reticulum vesicles was examined using the calcium indicator antipyrylazo III. Calcium release was initiated by addition of inositol 1,4,5-trisphosphate (IP₃) to aortic vesicles 7 min after initiation of ATP-supported calcium uptake. Half-maximal calcium release occurred at 1 μM IP₃, with maximal calcium release amounting to 25±2% of the intravesicular calcium (n=12, 9 preparations). Ruthenium red (10–20 μM), which has been reported to block IP₃-induced calcium release from skeletal muscle sarcoplasmic reticulum, did not inhibit aortic IP₃-induced calcium release. Elevation of Mg²⁺ concentration from 0.06 to 7.8 mM inhibited aortic IP₃-induced calcium release 75%, which contrasts with the Mg²⁺-insensitive IP₃-induced calcium release from platelet reticular membranes. The IP₃-dependence of aortic calcium release suggested that Mg²⁺ acted as a noncompetitive inhibitor. Thus, aortic sarcoplasmic reticulum vesicles contain an IP₃-sensitive calcium pathway which is inhibited by millimolar concentrations of Mg²⁺, but which is not inhibited by Ruthenium red and so differs from the previously described IP₃-sensitive calcium pathways in skeletal muscle and platelet reticular membranes.     

A Subset of Porcine Reproductive and Respiratory Syndrome Virus GP3 Glycoprotein Is Released into the Culture Medium of Cells as a Non-Virion-Associated and Membrane-Free (Soluble) Form

The GP₃ protein of the IAF-Klop strain of porcine reproductive and respiratory syndrome virus (PRRSV) was expressed in 293 cells by a recombinant human type 5 adenovirus carrying the open reading frame 3 gene. The protein exhibited a molecular mass of 42 kDa and comigrated with GP₃ expressed in PRRSV-infected MARC-145 cells. Removal of N-linked glycans from GP₃ resulted in a 27-kDa protein (P3), confirming its highly glycosylated nature. Pulse-chase experiments carried out either in the context of PRRSV infection or upon individual expression of GP₃ in 293 cells showed that the protein remains completely endo-β-N-acetylglucosaminidase H-sensitive even after 4 h of synthesis. Thus, the transport of GP₃ was restricted to the premedial Golgi compartment, presumably the endoplasmic reticulum (ER). However, a minor fraction of GP₃ was found to be secreted in the culture medium as a soluble membrane-free form. This released protein (sGP₃) was readily identified upon individual expression of GP₃ in 293 cells as well as in the context of PRRSV infection, albeit at lower levels. The sGP₃ migrated as a smear and displayed a molecular mass ranging from 43 to 53 kDa. The unglycosylated form of sGP₃ comigrated with its intracellular deglycosylated counterpart, suggesting that the release from the cell of a subset of GP₃ did not result from cleavage of a putative membrane-anchor sequence. Strikingly, unlike GP₃, the sGP₃ acquired Golgi-specific modifications of its carbohydrate side chains and folded into a disulfide-linked homodimer. Brefeldin A treatment completely abolished the release of sGP₃, suggesting that the ER-to-Golgi compartment is an obligatory step in cellular secretion of sGP₃. In contrast, 10 mM monensin did not prevent sGP₃ release but inhibited the terminal glycosylation that confers on the protein its diffuse pattern. Since GP₃ was found to be nonstructural in the case of the North American strain, secretion of a minor fraction of GP₃ might be an explanation for its high degree of immunogenicity in infected pigs. Furthermore, this secreted protein might be relevant as a model for further studies on the cellular subcompartments involved in the sorting of proteins to the extracellular milieu.     

Protein glycosylation regulates the externalization of two distinct classes of glucocorticoid-induced glycoproteins in rat hepatoma cells

The relationship of protein glycosylation to the externalization of glucocorticoid inducible alpha1-acid glycoprotein and mouse mammary tumor virus glycoproteins was examined in M1.54, a clonal population of mouse mammary tumor virus-infected rat hepatoma cells. Multiple freeze-thaw of isolated microsomes revealed that while alpha 1-acid glycoprotein is carried through the cell as a soluble component of vesicles, extracellular viral glycoproteins are initially membrane-associated. At concentrations of tunicamycin that specifically inhibited N-linked protein glycosylation, alpha 1-acid glycoprotein fractionated between the cellular and extracellular compartments. Thus, approximately one half of the newly synthesized, nonglycosylated (22,000 Mr) alpha 1-acid glycoprotein was rapidly secreted with kinetics similar to its glycosylated counterpart (release half-time of 60 min), while the remaining species first localized in an undefined intracellular compartment prior to its slow secretion (release half-time of 24 h). The same distribution of nonglycosylated alpha 1-acid glycoprotein was observed at various absolute levels of polypeptide, suggesting that this was not due simply to the saturation of an efficient secretory pathway at high polypeptide levels. In contrast to alpha 1-acid glycoprotein, no labeled viral antigens were released by tunicamycin-treated M1.54, while a nonglycosylated viral precursor glycopolyprotein was expressed intracellularly. Taken together, these results suggest that carbohydrate attachment strongly regulates the externalization of both alpha 1-acid glycoprotein and mouse mammary tumor virus species, which represent two distinct classes of extracellular glycoproteins.     

Lowering extracellular sodium or pH raises intracellular calcium in gastric cells

Summary The dependence of cytoplasmic free [Ca] (Ca _i ) on [Na] and pH was assessed in individual parietal cells of intact rabbit gastric glands by microfluorimetry of fura-2. Lowering extracellular [Na] (Na _o ) to 20mm or below caused a biphasic Ca _i increase which consisted of both release of intracellular Ca stores and Ca entry across the plasma membrane. The Ca increase was not blocked by antagonists of Ca-mobilizing receptors (atropine or cimetidine) and was independent of the replacement cation. Experiments in Ca-free media and in Na-depleted cells indicated that neither phase was due to reversal of Na/Ca exchange. The steep dependence of the Ca _i increase on Na _o suggested that the response was not due to lowering intracellular [Na] (Na _i ). The effects of low Na _o on Ca _i were also completely independent of changes in intracellular pH (pH _i ). Ca _i was remarkably stable during changes of pH _i of up to 2 pH units, indicating that H and Ca do not share a cytoplasmic buffer system. Such large pH excursions required determination of the pH dependence of fura-2. Because fura-2 was found to decrease its affinity for Ca as pH decreased below 6.7, corrections were applied to experiments in which large pH _i changes were observed. In contrast to the relative insensitivity of Ca _i to changes in pH _i , decreasing extracellular pH (pH _o ) to 6.0 or below was found to stimulate release of intracellular Ca stores. Increased Ca entry was not observed in this case. The ability of decreases in Na _o and pH _o to stimulate release of intracellular Ca stores suggest interactions between Na and H with extracellular receptors.     

Guinea Pig Histamine H1 Receptor. II. Stable Expression in Chinese Hamster Ovary Cells Reveals the Interaction with Three Major Signal Transduction Pathways

Abstract: A cDNA encoding a guinea pig histamine H₁ receptor was stably expressed in Chinese hamster ovary (CHO) cells. In one resulting clone, named CHO(H₁), the H₁ receptor was found to be coupled to several major signal transduction pathways. In each case the involvement of a G_i/G_o protein with pertussis toxin (PTX) was assessed, as well as the influence of extracellular Ca²⁺ and of protein kinase C activation by phorbol 12-myristate 13-acetate (PMA). Histamine induced, in a PTX- and PMA-insensitive manner, a biphasic increase in the intracellular Ca²⁺ level of which only the second sustained phase was dependent on the extracellular Ca²⁺ level. Histamine also caused a threefold elevation of inositol phosphate production, which was PTX-insensitive, but slightly inhibited by PMA and reduced by 75% in the absence of extracellular Ca²⁺. Histamine also caused a massive release of arachidonic acid, which occurred in a Ca²⁺- and PMA-sensitive manner, probably through the activation of a cytosolic phospholipase A₂, which partly involves coupling to a PTX-sensitive G protein. In comparison, in HeLa cells endowed with a native H₁ receptor, the histamine-induced arachidonic acid release was also Ca²⁺- and PMA-sensitive, but totally PTX-insensitive. Finally, in CHO(H₁) cells, histamine in very low concentrations potentiated the cyclic AMP accumulation induced by forskolin. This response appeared to be insensitive to PTX, extracellular Ca²⁺, and PMA. These various observations show that stimulation of a single receptor subtype, the guinea pig H₁ receptor, can trigger four major intracellular signals through coupling to several G proteins that are variously modulated by extracellular Ca²⁺ and protein kinase C activation.     

CO2/HCO3?- and Calcium-regulated Soluble Adenylyl Cyclase as a Physiological ATP Sensor

The second messenger molecule cAMP is integral for many physiological processes. In mammalian cells, cAMP can be generated from hormone- and G protein-regulated transmembrane adenylyl cyclases or via the widely expressed and structurally and biochemically distinct enzyme soluble adenylyl cyclase (sAC). sAC activity is uniquely stimulated by bicarbonate ions, and in cells, sAC functions as a physiological carbon dioxide, bicarbonate, and pH sensor. sAC activity is also stimulated by calcium, and its affinity for its substrate ATP suggests that it may be sensitive to physiologically relevant fluctuations in intracellular ATP. We demonstrate here that sAC can function as a cellular ATP sensor. In cells, sAC-generated cAMP reflects alterations in intracellular ATP that do not affect transmembrane AC-generated cAMP. In β cells of the pancreas, glucose metabolism generates ATP, which corresponds to an increase in cAMP, and we show here that sAC is responsible for an ATP-dependent cAMP increase. Glucose metabolism also elicits insulin secretion, and we further show that sAC is necessary for normal glucose-stimulated insulin secretion in vitro and in vivo.     

Direct and Indirect Effects of Mutations at the Outer Mouth of the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Pore

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel pore is thought to contain multiple binding sites for permeant and impermeant anions. Here, we investigate the effects of mutation of different positively charged residues in the pore on current inhibition by impermeant Pt(NO₂)₄²⁻ and suramin anions. We show that mutations that remove positive charges (K95, R303) influence interactions with intracellular, but not extracellular, Pt(NO₂)₄²⁻ ions, consistent with these residues being situated within the pore inner vestibule. In contrast, mutation of R334, supposedly located in the outer vestibule of the pore, affects block by both extracellular and intracellular Pt(NO₂)₄²⁻. Inhibition by extracellular Pt(NO₂)₄²⁻ requires a positive charge at position 334, consistent with a direct electrostatic interaction resulting in either open channel block or surface charge screening. In contrast, inhibition by intracellular Pt(NO₂)₄²⁻ is weakened in all R334-mutant forms of the channel studied, inconsistent with a direct interaction. Furthermore, mutation of R334 had similar effects on block by intracellular suramin, a large organic molecule that is apparently unable to enter deeply into the channel pore. Mutation of R334 altered interactions between intracellular Pt(NO₂)₄²⁻ and extracellular Cl⁻ but not those between intracellular Pt(NO₂)₄²⁻ and extracellular Pt(NO₂)₄²⁻. We propose that while the positive charge of R334 interacts directly with extracellular anions, mutation of this residue also alters interactions with intracellular anions by an indirect mechanism, due to mutation-induced conformational changes in the protein that are propagated some distance from the site of the mutation in the outer mouth of the pore.     

Calcium Dynamics During Physiological Acidification in Xenopus Oocyte

Interplays between intracellular pH (pHi) and calcium ([Ca²⁺]_i) variations remain unclear, though both proton and calcium homeostasis changes accompany physiological events such as Xenopus laevis oocyte maturation. In this report, we used NH₄Cl and changes of extracellular pH (pHe) to acidify the cytosol in a physiological range. In oocytes voltage-clamped at −80 mV, NH₄Cl triggered an inward current, the main component of which is a Ca²⁺-dependent chloride current. Calcium imaging confirmed that NH₄Cl provoked a [Ca²⁺]_i increase. The mobilized sources of calcium were discriminated using the triple-step protocol as a means to follow both the calcium-activated chloride currents (ICl-Ca) and the hyperpolarization- and acid-activated nonselective cation current (I_In). These currents were stimulated during external addition of NH₄Cl. This upregulation was abolished by BAPTA-AM, caffeine and heparin. By both buffering pHi changes with MOPS and by inhibiting calcium influx with lanthanum, intracellular acidification, initiated by NH₄Cl and extracellular acidic medium, was shown to trigger a [Ca²⁺]_i increase through both calcium release and calcium influx. The calcium pathways triggered by pHe changes are similar to those activated by NH₄Cl, thus suggesting that there is a robust signaling mechanism allowing the cell to adjust to variable environmental conditions.