ATP对巨噬细胞内吞和溶酶体pH的影响

ＦＩＴＣ－ｄｅｘｔｒａｎ标记培养的小鼠腹腔巨噬细胞溶酶体,ＣｏｎＡ－ＦＩＴＣ标记细胞内吞。用激光扫描共聚焦显微镜测量伴刀豆球蛋白（ＣｏｎＡ）、ＡＴＰ引起的巨噬细胞溶酶体ｐＨ动态变化和ＡＴＰ对细胞内吞ＣｏｎＡ－ＦＩＴＣ的影响。结果显示ＣｏｎＡ引起巨噬细胞溶酶体ｐＨ迅速增加,６ｍｉｎ左右达到峰值（ｐＨ５．７）;ＡＴＰ刺激３０ｍｉｎ后再加入ＣｏｎＡ,溶酶体ｐＨ无明显变化（ｐＨ４．０）;同时加入ＡＴＰ和ＣｏｎＡ,５ｍｉｎ左右溶酶体ｐＨ降到最低点（ｐＨ４．１）;ＡＴＰ对巨噬细胞内吞ＣｏｎＡ－ＦＩＴＣ有明显的抑制作用。探讨了受体介导内吞与溶酶体ｐＨ的关系。     

巨噬细胞吞噬过程中膜磷脂流动性和受体流动性的关系

本文用ＦＲＡＰ（ｆｌｕｏｒｅｓｃｅｎｃｅｒｅｃｏｖｅｒｙａｆｔｅｒｐｈｏｔｏｂｌｅａｃｈｉｎｇ）技术,测量了静息状态和刀豆素Ａ刺激不同时间后巨噬细胞膜磷脂、ＣｏｎＡ受体扩散系数和荧光恢复率的变化。结果显示ＣｏｎＡ刺激后膜磷脂和ＣｏｎＡ受体的扩散系数和荧光恢复率均较静息状态的巨噬细胞明显降低,磷脂流动性的变化与ＣｏｎＡ受体流动性的变化呈正相关。提示受体介导内吞导致的膜磷脂流动性的降低,可能是由于配体与细胞膜上受体结合形成配体－受体复合体,增加了受体的负荷,使受体的流动性降低,进而使膜磷脂的流动性降低。巨噬细胞内吞过程中膜磷脂和ＣｏｎＡ受体流动性的降低,可能还与ＣｏｎＡ刺激后巨噬细胞胞浆ｐＨ值有关。     

共聚焦镜观察凋亡巨噬细胞内pH的变化

用透射电镜观察巨噬细胞的形态学改变,结果显示,地塞米松处理８小时后,大部分巨噬细胞发生凋亡特征变化：胞突缩短、减少,胞膜完整。胞体皱缩,胞质密度增加,其中出现大量空泡。胞核染色质边聚、浓缩。另外用激光扫描共聚焦显微镜（ＡＣＡＳ５７０）和ｐＨ荧光探针ＳＮＡＲＦ┐１／ＡＭ实时检测地塞米松处理巨噬细胞胞浆ｐＨ的动态变化。加入地塞米松,多数巨噬细胞胞浆马上发生快速和短期的碱化。随后,胞浆ｐＨ缓慢降低,胞浆酸化。结果表明,胞浆酸化是细胞凋亡发展的必然过程,胞浆碱化则很可能与细胞凋亡的发生相关,也可能与细胞种类、细胞功能状态相关     

内溶酶体–自噬溶酶体对阿尔茨海默症病理进程的影响

自噬是在细胞受到胞内应激或饥饿条件下,依赖于溶酶体将胞内异常蛋白质以及受损细胞器降解的过程。内体是由细胞内吞形成的单层膜结构细胞器,它可以内吞进入细胞的异常蛋白质将其送入自噬体或通过内溶酶体–自噬溶酶体途径降解。由于自噬体与内体在形态与功能上相互联系又有相似之处,从而构成内溶酶体–自噬溶酶体系统。在阿尔茨海默症(Alzheimer’s disease,AD)患者的神经元中,两种异常蛋白质[β淀粉样物质(βamyloid,Aβ)和过度磷酸化的Tau蛋白]可以通过内溶酶体–自噬溶酶体系统清除;而当此系统功能受阻时,神经元中出现异常自噬体与内体形成的颗粒空泡变性体,导致AD病理改变加重。因此,内溶酶体–自噬溶酶体在阿尔茨海默病中扮演着重要角色。越来越多的研究结果提示,对内溶酶体–自噬溶酶体系统的调控可能为阿尔茨海默病的治疗提供新靶点和方向。     

单抗LC-1与针对的人肺腺癌SPC-A-1细胞肿瘤相关抗原复合物的内化研究

我们运用抗人肺癌单抗LC-1结合胶体金免疫电镜技术研究了人肺腺癌SPC-A-1细胞表面抗原抗体复合物被内吞的全过程,发现该细胞表面抗原抗体复合物是通过受体介导内吞途径被内化,经多泡体富集后至溶酶体消化降解。此外我们还用流式细胞仪分析了内吞前后该细胞表面抗原量的变化和短期内的恢复情况。LC-1在诱发该细胞表面抗原内化的同时还诱发了它对该核糖体的自噬。     

单抗LC—1与针对的人肺腺癌SPC—A—1细胞肿瘤相关抗原复合物的内…

我们运用抗人肺癌单抗ＬＣ－１结合胶体金免疫电镜技术研究了人肺腺癌ＳＰＣ－Ａ－１细胞表面抗原抗复合物复合物被内吞的全过程,发现该细胞表面抗原抗仨复合物是通过受体介导内吞途径被内化,经多泡体富集后至溶酶体消化降解。此外我们还用流式细胞仪分析了内吞前后该细胞表面抗原量的变化和短期内的恢复情况。ＬＣ－１在诱发该细胞表面抗原内化的同时还诱发了它对该核糖体的自噬。     

G_(M1)介导的体外培养神经元对CT-HRP的内吞

本实验用霍乱毒素辣根过氧化物酶复合物(CT-HRP)与培养的新生大鼠小脑细胞直接结合的电镜细胞化学方法,表明神经节苷脂G_M1只分布在细胞膜的外侧面;神经细胞表面的G_M1含量比胶质细胞高。通过电镜还观察到神经细胞和少突胶质细胞对结合在膜上的CT-HRP的不同内吞过程。培养的细胞在4℃与CT-HRP孵育1小时后,CT-HRP仅结合在细胞膜的外表面,无内吞现象出现。如将标本再移入36.5℃中温育,15分钟内即出现神经细胞对结合在膜上的CT-HRP的内吞,称为受体介导的细胞内吞(RME)。RME不仅发生在神经细胞的胞体部位,在突起上也多见。神经细胞RME的特点是包含G_M1-CT-HRP的内吞小泡必先进入与膜的再循环有关的GERL区。当温育延长至3小时,细胞膜上的结合CT-HRP的数量与温育初期无明显差异,内吞小泡仍在GERL区,并可见胞体和树突内若干阳性小泡成串地沿微管排列,在细胞膜内侧的胞浆内也有较多的阳性小泡分布。少突胶质细胞在温育15分钟内吞入大部分结合在膜上的CT-HRP,形成大量内吞小泡,弥散地分布在细胞质内。随着温育时间的延长,内吞小泡彼此融合,最终与溶酶体融合。本文就这两种细胞对CT- ??HRP的不同内吞的可能原因及其生物学意义进行了讨论。     

肽链的翻译后加工4.溶酶体酶的加工,分拣和投送

肽链的翻译后加工４．溶酶体酶的加工、分拣和投送王克夷（中国科学院上海生物化学研究所,上海２０００３１）关键词翻译后加工,溶酶体酶溶酶体是细胞内大分子降解的主要场所,有两个主要特征：一是它腔内的酸性ｐＨ;二是含有多达５０种的可溶性水解酶,包括蛋白水解酶...     

运动诱导的低铁状态大鼠骨髓细胞铁摄入的变化

本文观察了运动性低铁状态大鼠骨髓细胞转铁蛋白 (Tf)结合铁和非Tf结合铁摄入的变化。大鼠随机分为 6个月的运动组 (EG)和对照组 (SG)。SG平均每个幼红细胞Tf受体数为 890 15 0± 16 4849个 ,而在EG为 2 17536 0± 46 2 737个 (P <0 0 5 ) ,但受体的解离常数不受运动影响。EG中Tf的内吞平台和胞内铁聚积速度显著高于SG ,胞浆和胞内膜性成分中Tf结合铁和Fe(Ⅱ )摄入增加。EG的胞浆内Fe(Ⅱ )摄入的米氏常数值降低 ;细胞膜性成分中Fe(Ⅱ )摄入的最大速度增加。上述结果表明 ,运动不仅通过增加Tf受体的表达促进Tf结合铁的内吞 ,而且增强非Tf结合铁的内吞途径。尽管这些变化的机制尚不清楚 ,但它们有利于运动时血红素的合成     

多泡体形成过程的细胞化学研究

本实验用酶细胞化学和示踪细胞化学方法观察了睾丸间质细胞中多泡体的形成过程及其与溶酶体的关系。实验结果表明,睾丸间质细胞中多泡体的形成可分三个阶段:首先,一些含内吞物质的泡状结构进入高尔基体区域,与那里的小泡融合,形成内含少量小泡的前多泡体;然后,前多泡体互相融合,形成体积较大、基质电子密度低、内含小泡排列稀疏的低电子密度多泡体;最后,低电子密度多泡体通过表面长出微绒毛样结构并不断断裂的方式去除多余的界膜,形成体积较小、基质电子密度高、内含小泡排列紧密的高电子密度多泡体。因此,多泡体的形成既与内吞活动有关,又与高尔基体区域小泡有关。前多泡体和低电子密度多泡体不含溶酶体酶。在多泡体形成过程中,只有到高电子密度多泡体阶段,才与溶酶体发生关系,从溶酶体中获取溶酶体酶。多泡体形成后,常与自体吞噬泡靠近,可能参与睾丸间质细胞的自体吞噬活动。     

受体介导内吞引起的巨噬细胞胞质酸化和胞吐作用

本文利用激光扫描共聚焦显微镜A-CAS570从细胞形态学和功能两方面,研究了刀豆素A(Concanavalin A,Con A)、麦芽凝集素(Wheat Germ Agglutinin,WGA)、酵母多糖(Zymosan A,Z.A)对小鼠腹腔巨噬细胞胞质pH和溶酶体内荧光探针FITC—Dextran排出细胞的影响。结果显示三种配体加入细胞外液10min内,胞质pH很快下降,此后维持在该水平;在15min左右细胞外FITC一Dextran迅速增加,20min后变化趋于停止;在三种配体加入后15min左右,细胞内溶酶体在质膜内侧增多;25—30min溶酶体重新向细胞中央运动。根据上述实验结果,我们认为溶酶体pH升高是触发溶酶体内荧光探针通过胞吐作用排出细胞的必要条件,胞质酸化抑制溶酶体内容物通过胞吐作用排出细胞。配体刺激引起的溶酶体内容物通过胞吐作用排出细胞和胞质酸化是细胞自我调节和保护的一种反映。     

Cytosolic acidification and lysosomal alkalinization during TNF-α induced apoptosis in U937 cells

Apoptosis is often associated with acidification of the cytosol and since loss of lysosomal proton gradient and release of lysosomal content are early events during apoptosis, we investigated if the lysosomal compartment could contribute to cytosolic acidification. After exposure of U937 cells to tumor necrosis factor-α, three populations; healthy, pre-apoptotic, and apoptotic cells, were identified by flow cytometry. These populations were investigated regarding intra-cellular pH and apoptosis-associated events. There was a drop in cytosolic pH from 7.2 ± 0.1 in healthy cells to 6.8 ± 0.1 in pre-apoptotic, caspase-negative cells. In apoptotic, caspase-positive cells, the pH was further decreased to 5.7 ± 0.04. The cytosolic acidification was not affected by addition of specific inhibitors towards caspases or the mitochondrial F₀F₁-ATPase. In parallel to the cytosolic acidification, a rise in lysosomal pH from 4.3 ± 0.3, in the healthy population, to 4.8 ± 0.3 and 5.5 ± 0.3 in the pre-apoptotic- and apoptotic populations, respectively, was detected. In addition, lysosomal membrane permeability increased as detected as release of cathepsin D from lysosomes to the cytosol in pre-apoptotic and apoptotic cells. We, thus, suggest that lysosomal proton release is the cause of the cytosolic acidification of U937 cells exposed to TNF-α.     

受体介导内吞引起的巨噬细胞胞质酸化和胞吐作用

The effects of Con A, WGA, Zymosan A on macrophage cytosolic pH and outflow of lysosomal content through exocytosis were studied with SNAFL-calcein and FITC-Dextran on ACAS570. The results showed all three ligands could induce macrophage cytosolic acidification in about 10 min and kept at the same level hereafter; outflow of lysosomal fluorescent probe through exocytosis appeared in 15-20 min. In resting conditions, macrophage lysosomes mainly distributed in cell center; after stimulated for 15 min by three ligands, the number of lysosomes increased in membrane periphery, in 25-30 min lysosomes moved back toward cell center. We proposed that ligands induced lysosomal pH rises was a basic factor for outflow of lysosomal content through exocytosis, cytosolic acidification inhibited receptor-mediated endocytosis. Cytosolic acidification and outflow of lysosomal content through exocytosis were the results of cellular self-regulation and self-protection during receptor-mediated endocytosis.     

Role of lysosomal and cytosolic pH in the regulation of macrophage lysosomal enzyme secretion.

Rapid and parallel secretion of lysosomal beta-N-acetylglucosaminidase and preloaded fluorescein-labelled dextran was initiated in macrophages by agents affecting intracellular pH (methylamine, chlorpromazine, and the ionophores monensin and nigericin). In order to evaluate the relative role of changes in lysosomal and cytosolic pH, these parameters were monitored by using pH-sensitive fluorescent probes [fluorescein-labelled dextran or 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein]. All agents except chlorpromazine caused large increases in lysosomal pH under conditions where they induced secretion. By varying extracellular pH and ion composition, the changes in lysosomal and cytosolic pH could be dissociated. Secretion was then found to be significantly modulated by changes in cytosolic pH, being enhanced by alkalinization and severely inhibited by cytosolic acidification. However, changes in cytosolic pH in the absence of stimulus were unable to initiate secretion. Dissociation of the effects on lysosomal and cytosolic pH was also achieved by combining stimuli with either nigericin or acetate. Further support for a role of intracellular pH in the control of lysosomal enzyme secretion was provided by experiments where bicarbonate was included in the medium. The present study demonstrates that an increase in lysosomal pH is sufficient to initiate lysosomal enzyme secretion in macrophages and provides evidence for a significant regulatory role of cytosolic pH.     

Consequences of loss of Vph1 protein-containing vacuolar ATPases (V-ATPases) for overall cellular pH homeostasis

In yeast cells, subunit a of the vacuolar proton pump (V-ATPase) is encoded by two organelle-specific isoforms, VPH1 and STV1. V-ATPases containing Vph1 and Stv1 localize predominantly to the vacuole and the Golgi apparatus/endosomes, respectively. Ratiometric measurements of vacuolar pH confirm that loss of STV1 has little effect on vacuolar pH. Loss of VPH1 results in vacuolar alkalinization that is even more rapid and pronounced than in vma mutants, which lack all V-ATPase activity. Cytosolic pH responses to glucose addition in the vph1Δ mutant are similar to those in vma mutants. The extended cytosolic acidification in these mutants arises from reduced activity of the plasma membrane proton pump, Pma1p. Pma1p is mislocalized in vma mutants but remains at the plasma membrane in both vph1Δ and stv1Δ mutants, suggesting multiple mechanisms for limiting Pma1 activity when organelle acidification is compromised. pH measurements in early prevacuolar compartments via a pHluorin fusion to the Golgi protein Gef1 demonstrate that pH responses of these compartments parallel cytosolic pH changes. Surprisingly, these compartments remain acidic even in the absence of V-ATPase function, possibly as a result of cytosolic acidification. These results emphasize that loss of a single subunit isoform may have effects far beyond the organelle where it resides.     

Role of vacuolar adenosine triphosphatase in the regulation of cytosolic pH in hepatocytes

The responses of the cytosolic pH of hepatocytes in suspension to agents affecting the activity of vacuolar adenosine triphosphatase (V-ATPase) and Na/H exchange have been studied. Changes of cytosolic pH were determined both with dual-wavelength excitation (500/440 nm) of the fluorescence of 2,7-bis-(2-carboxyethyl)-5(and 6)-carboxyfluorescein and from the distribution of ¹⁴C-dimethyloxazolidinedione; both methods gave very similar results. Changes of vesicular pH were determined by comparing the fluorescence of fluorescein isothiocyanate-dextran and rhodamine B isothiocyanate-dextran taken up by endocytosis. Nitrate, which inhibits V-ATPase in isolated organelles, induced a concentration-dependent acidification of the cytosol and alkalinization of vesicles, with maximal effects at 25–37.5 mm in each case, indicating that V-ATPase contributes to removal of cytosolic protons. On continued exposure to nitrate, the acidification underwent an amiloride-inhibitable reversal. At the higher concentrations of NO ₃ ^– , both cytosolic acidification and vesicular alkalinization were reduced or absent. Bafilomycin A₁ caused alkalinization of vesicular pH; cytosolic acidification was not observed, possibly because of other ionic exchanges. Recovery of cytosolic pH from an acid load (2 min exposure to 5% CO₂) was sensitive to both 25 mm NO ₃ ^– and to ouabain. The pH dependence of the nitrate effect was tested with media of different pH; the activity was negligible at cytosolic pH 6.2 and rose to a maximum at cytosolic pH 7.3. Treatment of hepatocytes with 0.5–1.0 mm ouabain resulted in an initial alkalinization (0.5–2 min duration) of the cytosol, followed by a spontaneous reversal and, on occasion, further acidification. The alkalinization was blocked by 25 mm NO ₃ ^– , but not by 25 mm gluconate. The results suggest that the cytosolic alkalinization is caused by a stimulation of H⁺ uptake by V-ATPase activity. We conclude that V-ATPases make an important contribution to the regulation of the cytosolic pH of hepatocytes.This work was supported in part by National Institutes of Health B.R.S. Grant 507 RR05417 to Temple University.     

Luminal and cytosolic pH feedback on proton pump activity and ATP affinity of V-type ATPase from Arabidopsis

Proton pumping of the vacuolar-type H(+)-ATPase into the lumen of the central plant organelle generates a proton gradient of often 1-2 pH units or more. Although structural aspects of the V-type ATPase have been studied in great detail, the question of whether and how the proton pump action is controlled by the proton concentration on both sides of the membrane is not understood. Applying the patch clamp technique to isolated vacuoles from Arabidopsis mesophyll cells in the whole-vacuole mode, we studied the response of the V-ATPase to protons, voltage, and ATP. Current-voltage relationships at different luminal pH values indicated decreasing coupling ratios with acidification. A detailed study of ATP-dependent H(+)-pump currents at a variety of different pH conditions showed a complex regulation of V-ATPase activity by both cytosolic and vacuolar pH. At cytosolic pH 7.5, vacuolar pH changes had relative little effects. Yet, at cytosolic pH 5.5, a 100-fold increase in vacuolar proton concentration resulted in a 70-fold increase of the affinity for ATP binding on the cytosolic side. Changes in pH on either side of the membrane seem to be transferred by the V-ATPase to the other side. A mathematical model was developed that indicates a feedback of proton concentration on peak H(+) current amplitude (v(max)) and ATP consumption (K(m)) of the V-ATPase. It proposes that for efficient V-ATPase function dissociation of transported protons from the pump protein might become higher with increasing pH. This feature results in an optimization of H(+) pumping by the V-ATPase according to existing H(+) concentrations.     

Vacuolar and plasma membrane proton pumps collaborate to achieve cytosolic pH homeostasis in yeast

Vacuolar proton-translocating ATPases (V-ATPases) play a central role in organelle acidification in all eukaryotic cells. To address the role of the yeast V-ATPase in vacuolar and cytosolic pH homeostasis, ratiometric pH-sensitive fluorophores specific for the vacuole or cytosol were introduced into wild-type cells and vma mutants, which lack V-ATPase subunits. Transiently glucose-deprived wild-type cells respond to glucose addition with vacuolar acidification and cytosolic alkalinization, and subsequent addition of K(+) ion increases the pH of both the vacuole and cytosol. In contrast, glucose addition results in an increase in vacuolar pH in both vma mutants and wild-type cells treated with the V-ATPase inhibitor concanamycin A. Cytosolic pH homeostasis is also significantly perturbed in the vma mutants. Even at extracellular pH 5, conditions optimal for their growth, cytosolic pH was much lower, and response to glucose was smaller in the mutants. In plasma membrane fractions from the vma mutants, activity of the plasma membrane proton pump, Pma1p, was 65-75% lower than in fractions from wild-type cells. Immunofluorescence microscopy confirmed decreased levels of plasma membrane Pma1p and increased Pma1p at the vacuole and other compartments in the mutants. Pma1p was not mislocalized in concanamycin-treated cells, but a significant reduction in cytosolic pH under all conditions was still observed. We propose that short-term, V-ATPase activity is essential for both vacuolar acidification in response to glucose metabolism and for efficient cytosolic pH homeostasis, and long-term, V-ATPases are important for stable localization of Pma1p at the plasma membrane.     

Cytosolic pH regulation in osteoblasts. Regulation of anion exchange by intracellular pH and Ca2+ ions

Measurements of cytosolic pH (pHi) 36Cl fluxes and free cytosolic Ca2+ concentration ([Ca2+]i) were performed in the clonal osteosarcoma cell line UMR-106 to characterize the kinetic properties of Cl-/HCO3- (OH-) exchange and its regulation by pHi and [Ca2+]i. Suspending cells in Cl(-)-free medium resulted in rapid cytosolic alkalinization from pHi 7.05 to approximately 7.42. Subsequently, the cytosol acidified to pHi 7.31. Extracellular HCO3- increased the rate and extent of cytosolic alkalinization and prevented the secondary acidification. Suspending alkalinized and Cl(-)-depleted cells in Cl(-)-containing solutions resulted in cytosolic acidification. All these pHi changes were inhibited by 4',4',-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) and H2DIDS, and were not affected by manipulation of the membrane potential. The pattern of extracellular Cl- dependency of the exchange process suggests that Cl- ions interact with a single saturable external site and HCO3- (OH-) complete with Cl- for binding to this site. The dependencies of both net anion exchange and Cl- self-exchange fluxes on pHi did not follow simple saturation kinetics. These findings suggest that the anion exchanger is regulated by intracellular HCO3- (OH-). A rise in [Ca2+]i, whether induced by stimulation of protein kinase C-activated Ca2+ channels, Ca2+ ionophore, or depolarization of the plasma membrane, resulted in cytosolic acidification with subsequent recovery from acidification. The Ca2+-activated acidification required the presence of Cl- in the medium, could be blocked by DIDS, and H2DIDS and was independent of the membrane potential. The subsequent recovery from acidification was absolutely dependent on the initial acidification, required the presence of Na+ in the medium, and was blocked by amiloride. Activation of protein kinase C without a change in [Ca2+]i did not alter pHi. Likewise, in H2DIDS-treated cells and in the absence of Cl-, an increase in [Ca2+]i did not activate the Na+/H+ exchanger in UMR-106 cells. These findings indicate that an increase in [Ca2+]i was sufficient to activate the Cl-/HCO3- exchanger, which results in the acidification of the cytosol. The accumulated H+ in the cytosol activated the Na+/H+ exchanger. Kinetic analysis of the anion exchange showed that at saturating intracellular OH-, a [Ca2+]i increase did not modify the properties of the extracellular site. A rise in [Ca2+]i increased the apparent affinity for intracellular OH- (or HCO3-) of both net anion and Cl- self exchange. These results indicate that [Ca2+]i modifies the interaction of intracellular OH- (or HCO3-) with the proposed regulatory site of the anion exchanger in UMR-106 cells.     

Caloric restriction controls stationary phase survival through Protein Kinase A (PKA) and cytosolic pH

Calorie restriction is the only physiological intervention that extends lifespan throughout all kingdoms of life. In the budding yeast Saccharomyces cerevisiae, cytosolic pH (pH_c) controls growth and responds to nutrient availability, decreasing upon glucose depletion. We investigated the interactions between glucose availability, pH_c and the central nutrient signalling cAMP‐Protein Kinase A (PKA) pathway. Glucose abundance during the growth phase enhanced acidification upon glucose depletion, via modulation of PKA activity. This actively controlled reduction in starvation pH_c correlated with reduced stationary phase survival. Whereas changes in PKA activity affected both acidification and survival, targeted manipulation of starvation pH_c showed that cytosolic acidification was downstream of PKA and the causal agent of the reduced chronological lifespan. Thus, caloric restriction controls stationary phase survival through PKA and cytosolic pH.