Cellular Distribution of Gangliosides in the Developing Mouse Cerebellum: Analysis Using the Staggerer Mutant

The distribution of cerebellar gangliosides was studied in staggerer (sg/sg) mutant mice, where the majority of granule cells die after completing their migration across the molecular layer. In addition, the external granule cell layer in sg/sg mice persists longer than in normal mice. Moreover, in the sg/sg cerebellum, Purkinje cells are significantly reduced in number, and almost none have tertiary branchlet spines. The loss of Purkinje cells and granule cells in sg/sg mice is accompanied by an early-onset reactive gliosis that continues through adulthood. By correlating changes in ganglioside composition with the well-documented histological events of cerebellar development in normal and sg/sg mice, we obtained strong evidence for a nonrandom cellular distribution of gangliosides. The sharpest reduction in the GD1a content of sg/sg cerebellum occurred after 15 days of age, coincident with granule cell loss. GT1a, on the other hand, was significantly reduced from 15 through 150 days in the sg/sg mice. GD3 is a major ganglioside of the undifferentiated granule cell, but it becomes rapidly displaced by the more complex gangliosides with the onset of granule cell maturation. In the sg/sg mice, GD3 persisted at abnormally high levels from 15 to 28 days and then accumulated through adulthood. These findings, and those from other cerebellar mouse mutants, suggest that GD1a is enriched in granule cells and that GT1a is enriched in Purkinje cells. Our findings also suggest that GT1a is more concentrated in branchlet spines than in other regions of the Purkinje cell membrane. GT1b appears to be enriched in both granule cells and Purkinje cells, whereas GM1 appears to be enriched in myelin. Furthermore, the apparent persistence of the embryonic ganglioside GD3 in sg/sg mice results from an early-onset reactive gliosis, together with a partial retardation in granule cell maturation. The accumulation of GD3 beyond 28 days reflects the continued accretion of GD3 in reactive glia.     

Evidence for a Hyperexcitability State of Staggerer Mutant Mice Macrophages

We recently reported an abnormal production of interleukin-1 (IL-1) in peripheral macrophages of several neurological mutant mice that exhibit patterns of neuronal degeneration, especially in the cerebellum. After in vitro activation by lipopolysaccharide acid (LPS), these macrophages hyperexpress IL-1 beta mRNA and hyperproduce IL-1 protein in comparison with +/+ controls. In the present study, focused on the staggerer mutant mice, we investigate if this genetic dysregulation is specific for IL-1 beta or if it reflects a generalized hyperexcitability of these macrophages. The hyperexpression of IL-1 beta mRNA in sg/sg macrophages is present whatever the duration of LPS stimulation, even for periods as short as 15 min, although it reaches a maximum after 4 h of stimulation. The hyperinducibility of sg/sg macrophages is observed even when very low doses of LPS are used (0.01 microgram/ml) and reaches its maximum for 5 micrograms/ml LPS. Synthetic molecules (muramyl dipeptides), such as N-acetylmuramyl-L-alanyl-D-isoglutamine or murabutide, known as macrophage activators, are also efficient in revealing the cytokine hyperexpression in sg/sg macrophages. In addition, hyperexpression of two other cytokines, i.e., tumor necrosis factor-alpha and IL-1 alpha mRNAs, is also detected in LPS-stimulated macrophages of mutant mice. Finally, the effect of an inhibitor of protein synthesis, cycloheximide, is similar in +/+ and sg/sg macrophages. As a whole, these data lead us to conclude that the sg/sg macrophages are in a state of general hyperexcitability when compared with +/+ ones.     

Changes in Particulate Neuraminidase Activity During Normal and Staggerer Mutant Mouse Development

Abstract: The activity of particulate neuraminidase (sialidase, EC 3.2.1.18) in wild-type mice and the neurological mutant Staggerer was studied during development. Peak activity of this enzyme was observed at postnatal day 3 (P3) in three tissues of normal mice: cerebellum, cerebrum, and liver. In Staggerer, however, neuraminidase peak activity was observed at P27 in the cerebellum, whereas the activity was close to normal in Staggerer cerebrum and liver. Activities of other glycosidases in Staggerer (α-glucosidase (pH 3.7), α- glucosidase (pH 6.0), N -acetyl-β-hexosaminidase, β-glucosidase, and β-galactosidase) did not show significant variation compared with wild-type at P27 in any of the three tissues. This indicates that the late activity peak of particulate neuraminidase activity in the Staggerer cerebellum is neuraminidase-specific and not due to a general increase of lysosomal enzymes.     

Thyroxine Injections Do Not Cause Premature Induction of Thymidine Kinase in sg/sg Mice

Severe hyperthyroidism from the time of birth causes a premature induction and termination of thymidine kinase activity in the cerebella of wild-type mice. This leads to elevated enzyme levels at postnatal days 5 and 6, with significantly lower levels by postnatal day 7 (which is actually the time of peak activity in normal animals). In this study, neonatal hyperthyroidism does not have significant effects on postnatal day 5, 6, or 7 enzyme levels in the neurological mutant staggerer. This is consistent with the hypothesis that thyroid hormone exerts its effects via the Purkinje cells, which are reduced in number and grossly stunted in the mutant.     

3Development-Dependent Regulation of N-Acetyl-β-d-Hexosaminidase of Cerebellum and Cerebrum of Normal and Staggerer Mutant Mice

Distinctive activities of various glycosidases were expressed in the cerebellum and cerebral cortex of mice during their development. In particular, N-acetyl-beta-D-hexosaminidase (EC 3.2.1.30) appeared to be developmentally regulated. A transient peak of enzyme activity at postnatal day 7 was characteristic for the cerebellum, whereas the activity in the cerebral cortex gradually increased through the 1st postnatal month and was maintained at a high level of activity throughout adulthood. The regulation of N-acetylhexosaminidase activity in the developing cerebellum of the staggerer mouse deviated clearly from enzyme activities in the wild-type, whereas the activity pattern in the staggerer cerebral cortex remained unaffected. In experiments mixing wild-type and staggerer cerebellum homogenates, the specific activity was additive. Thus, involvement of inhibitors or activating molecules can be excluded. This developmentally controlled regulation or disregulation in staggerer appears to be enzyme specific, sine beta-glucosidase, alpha-glucosidase, and beta-galactosidase did not exhibit such a pattern in either normal or staggerer mice. In the mutation weaver that, like staggerer, loses the majority of its cerebellar granule cells, N-acetyl-beta-hexosaminidase activity of the cerebellum was not elevated, indicating a specific defect in staggerer rather than a general effect on lysosomal enzymes due to cell death.     

慢性酒精中毒对成年小鼠小脑浦肯野细胞超微结构的影响

目的观察慢性酒精中毒所致的成年小鼠小脑皮质浦肯野细胞(Purkinje cell,PC)胞体的超微结构变化,探讨其对神经元超微结构的影响方式及生理意义。方法用15%酒精饲喂3月龄小白鼠3个月,经行为学检测后,取小脑前叶做电镜包埋,切片,染色,透射电镜下观察并拍照。结果酒精中毒组PC核周质中线粒体膨解,基质囊泡化;高尔基复合体扁平囊扩张;粗面内质网碎裂,核糖体颗粒减少;空泡变性出现;双层核膜界限不清;染色质边集等变化。结论慢性酒精中毒可导致小脑浦肯野细胞多种细胞器出现异常改变,推测这些变化可引起胞内物质合成减少,空间构筑紊乱,神经元死亡,最终导致小脑功能损伤。     

Sphingolipid Biosynthesis Is Necessary for Dendrite Growth and Survival of Cerebellar Purkinje Cells in Culture

Abstract: The requirement of complex sphingolipid biosynthesis for growth of neurons was examined in developing rat cerebellar Purkinje neurons using a dissociated culture system. Purkinje cells developed well-differentiated dendrites and axons after 2 weeks in a serum-free nutrient condition. Addition of 2 µM fumonisin B₁, a fungal inhibitor of mammalian ceramide synthase, inhibited incorporation of [³H]galactose/glucosamine and [¹⁴C]serine into complex sphingolipids of cultured cerebellar neurons. Under this condition, the expression of Purkinje cell-enriched sphingolipids, including GD1α, 9-O-acetylated LD1 and GD3, and sphingomyelin, was significantly decreased. After 2 weeks' exposure to fumonisin B₁, dose-dependent measurable decreases in the survival and visually discernible differences in the morphology were seen in fumonisin-treated Purkinje cells. The Purkinje cell dendrites exhibited two types of anomalies; one population of cells developed elongated but less-branched dendrites after a slight time lag, but their branches began to degenerate. In some cells, formation of elongated dendrite trees was severely impaired. However, treatment with fumonisin B₁ also led to the formation of spinelike protrusions on the dendrites of Purkinje cells as in control cultures. In contrast to the alterations observed in Purkinje cells, morphology of other cell types including granule neurons appeared to be almost normal after treatment with fumonisin B₁. These observations indicated strongly that membrane sphingolipids participate in growth and maintenance of dendrites and in the survival of cerebellar Purkinje cells. Indeed, these effects of fumonisin B₁ were reversed, but not completely, by the addition of 6-[[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]caproyl]sphingosine (C₆-NBD-ceramide), a synthetic derivative of ceramide. Thus, we conclude that deprivation of membrane sphingolipids in a culture environment is responsible for aberrant growth of Purkinje cells.     

CFTR Channels Expressed in CHO Cells Do Not Have Detectable ATP Conductance

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated, ATP-dependent chloride channel which may have additional functions. Recent reports that CFTR mediates substantial electrodiffusion of ATP from epithelial cells have led to the proposal that CFTR regulates other ion channels through an autocrine mechanism involving ATP. The aim of this study was to determine the ATP conductance of wild-type CFTR channels stably expressed in Chinese hamster ovary cells using patch clamp techniques. In the cell-attached configuration with 100 mm Mg · ATP or Tris · ATP solution in the pipette and 140 mm NaCl in the bath, exposing cells to forskolin caused the activation of a low-conductance channel having kinetics resembling those of CFTR. Single channel currents were negative at the resting membrane potential (V _m ), consistent with net diffusion of Cl from the cell into the pipette. The transitions decreased in amplitude, but did not reverse direction, as V _m was clamped at increasingly positive potentials to enhance the driving force for inward ATP flow (>+80 mV). In excised patches, single channel currents did not reverse under essentially biionic conditions (Cl_in/ATP_out or ATP_in/Cl_out), although PKA-activated currents were clearly visible in the same patches at voltages where they would be carried by chloride ions. Moreover, with NaCl solution in the bath and a mixture of ATP and Cl in the pipette, the single channel I/V curve reversed at the predicted equilibrium potential for chloride. CFTR channel currents disappeared when patches were exposed to symmetrical ATP solutions and were restored by reexposure to Cl solution. Finally, in the whole-cell configuration with NaCl in the bath and 100 mm MgATP or TrisATP in the pipette, cAMP-stimulated cells had time-independent, outwardly rectifying currents consistent with CFTR selectivity for external Cl over internal ATP. Whole-cell currents reversed near V _m =−55 mV under these conditions, however the whole cell resistance measured at −100 mV was comparable to that of the gigaohm seal between the plasma membrane and glass pipette (7 GΩ). We conclude that CFTR does not mediate detectable electrodiffusion of ATP. Received: 8 November 1995/Revised: 23 January 1996     

Abnormal cerebellar development and Purkinje cell defects in Lgl1-Pax2 conditional knockout mice

Lgl1 was initially identified as a tumour suppressor in flies and is characterised as a key regulator of epithelial polarity and asymmetric cell division. A previous study indicated that More-Cre-mediated Lgl1 knockout mice exhibited significant brain dysplasia and died within 24 h after birth. To overcome early neonatal lethality, we generated Lgl1 conditional knockout mice mediated by Pax2-Cre, which is expressed in almost all cells in the cerebellum, and we examined the functions of Lgl1 in the cerebellum. Impaired motor coordination was detected in the mutant mice. Consistent with this abnormal behaviour, homozygous mice possessed a smaller cerebellum with fewer lobes, reduced granule precursor cell (GPC) proliferation, decreased Purkinje cell (PC) quantity and dendritic dysplasia. Loss of Lgl1 in the cerebellum led to hyperproliferation and impaired differentiation of neural progenitors in ventricular zone. Based on the TUNEL assay, we observed increased apoptosis in the cerebellum of mutant mice. We proposed that impaired differentiation and increased apoptosis may contribute to decreased PC quantity. To clarify the effect of Lgl1 on cerebellar granule cells, we used Math1-Cre to specifically delete Lgl1 in granule cells. Interestingly, the Lgl1-Math1 conditional knockout mice exhibited normal proliferation of GPCs and cerebellar development. Thus, we speculated that the reduction in the proliferation of GPCs in Lgl1-Pax2 conditional knockout mice may be secondary to the decreased number of PCs, which secrete the mitogenic factor Sonic hedgehog to regulate GPC proliferation. Taken together, these findings suggest that Lgl1 plays a key role in cerebellar development and folia formation by regulating the development of PCs.     

Biosynthesis, mode of action and functional significance of neurosteroids in the developing Purkinje cell

New findings over the past decade have shown that the brain has the capability of forming steroids de novo from cholesterol, the so-called “neurosteroids”. To understand neurosteroid action in the brain, data on the regio- and temporal-specific synthesis of neurosteroids are needed. Recently, we have demonstrated that the Purkinje cell, a cerebellar neuron, is a major site for neurosteroid formation in a variety of vertebrates. This is the first demonstration of de novo neuronal neurosteroidogenesis in the brain. Since this discovery, organizing actions of neurosteroids are becoming clear by the studies on mammals using the Purkinje cell as an excellent cellular model. In mammals, the Purkinje cell actively synthesizes progesterone de novo from cholesterol during neonatal life, when cerebellar neuronal circuit formation occurs. The Purkinje cell may also produces estradiol in the neonate. Interestingly, both progesterone and estradiol promote dendritic growth, spinogenesis and synaptogenesis via each cognate nuclear receptor in the developing Purkinje cell. Such organizing actions may contribute to the formation of cerebellar neuronal circuit during neonatal life. This paper summarizes the advances made in our understanding of the biosynthesis, mode of action and functional significance of neurosteroids in the developing Purkinje cell.     

Ceramide and Its Interconvertible Metabolite Sphingosine Function as Indispensable Lipid Factors Involved in Survival and Dendritic Differentiation of Cerebellar Purkinje Cells

Abstract: Ceramide generated from sphingomyelin has emerged as a new but conserved type of biologically active lipid. We previously found that endogenous sphingolipids are required for the normal growth of cultured cerebellar Purkinje neurons and that sphingomyelin is present abundantly in the somatodendritic region of these cells. To gain further insight into a potential role of the sphingomyelin/ceramide pathway, we investigated the effects of depletion of sphingolipids on the phenotypic growth and survival of immature Purkinje cells and the ability of ceramide or other sphingolipids to antagonize these effects. Inhibition of ceramide synthesis by ISP-1, a specific inhibitor of serine palmitoyltransferase, decreased cellular levels of sphingolipids. This treatment resulted in a decrease in cell survival accompanied by an induction of apoptotic cell death and aberrant dendritic differentiation of Purkinje cells with no detectable changes in other cerebellar neurons. Cell-permeable ceramides, sphingosine, or sphingomyelin overcame these abnormalities more effectively than other sphingolipids when added simultaneously with ISP-1. Exposure to bacterial sphingomyelinase in turn enhanced cell survival and dendritic branching complexity of Purkinje cells at different optimal concentrations. Furthermore, cell-permeable ceramide acted synergistically with the neurotrophin family, which has been previously shown to support Purkinje cell survival. These observations suggest that ceramide is a requisite for the survival and the dendritic differentiation of Purkinje cells.     

小脑浦肯野细胞动作电位传输保真度在大鼠出生后发育期间的变化

小脑浦肯野细胞的轴突是小脑皮层唯一的传出通路,研究其动作电位传输能力对于了解小脑的生理功能具有重要意义.大鼠在出生后第2周到第3周,小脑浦肯野细胞的形态及功能都有显著变化,产生动作电位的能力明显增强.而轴突上动作电位传输能力的变化还有待研究.运用胞体以及轴突的双电极膜片钳技术,研究了出生8天以及15天的Wistar大鼠小脑浦肯野细胞轴突上动作电位的传输.与8天组相比,15天组大鼠小脑浦肯野细胞轴突上动作电位的传输能力明显增强.后超极化可以增强8天组轴突上动作电位的传输能力.研究表明,随着发育的成熟,动作电位的产生能力以及轴突上动作电位的传输能力同步增强.     

Presence of Calmodulin and Calmodulin-Binding Proteins in the Nuclei of Brain Cells

The nuclear calmodulin levels have been measured in rat neurons and glial cells. The values are 1.0 and 1.1 γg/ mg of protein, respectively. These levels are about threefold higher than those in the nuclei of rat liver cells. We have also investigated the presence of several calmodulin-binding proteins in the nuclei of both brain cellular types. As similarly observed in the nuclei of liver cells, we detected the presence of a-spectrin and a 62-kDa calmodulin-binding protein (p62) in the nuclei of neurons and glial cells by irnmunoblotting and immunocytochemical methods. Both proteins are enriched in the purified nuclear matrix samples from both cellular types. In contrast to that occurring in rat hepatocytes, we have not been able to detect, by irnmunoblotting methods, caldesmon in the nuclear matrices of neurons and glial cells. The immunocytochemical studies suggest, however, that caldesmon can be present in the nuclei but in a fraction distinct from the nuclear matrices.     

Highly Sensitive Immunoassay for Rat Brain-Type Creatine Kinase: Determination in Isolated Purkinje Cells

Ultrasensitive enzyme immunoassay method for the measurement of rat brain-type creatine kinase BB (CK-BB) was developed by use of purified antibodies specific to the B subunit of creatine kinase. The antibody immunoglobulin G was purified with immunoaffinity chromatography of the antiserum raised in rabbits by injecting the purified rat CK-BB. The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli. The assay was specific to the B subunit of CK (CK-B), showing about 10% cross-reactivity with CK-MB, but it did not cross-react with CK-MM and neuron-specific gamma gamma enolase. The minimum detection limit of the assay was 0.1 pg or 1 amol CK-BB, being sufficiently sensitive for the measurement of CK-B contents in the isolated Purkinje cell bodies at the level of single cells. The average content of CK-B in a single Purkinje cell was 1.64 pg. The CK-B concentration in rat cerebellum (about 22 micrograms/mg protein) was about twofold higher than that (about 13 micrograms/mg protein) in the cerebrum. High levels (greater than 5 micrograms/mg protein) of CK-B were also found in the peripheral tissues such as gastrointestinal tract and urinary bladder, all of which are composed of smooth muscle. Immunohistochemical localization of CK-B antigens in the CNS revealed that the antigens is distributed not only in the neurons but also in the glial cells.     

Purification and Characterization of P400 Protein, a Glycoprotein Characteristic of Purkinje Cell, from Mouse Cerebellum

P400 protein is a concanavalin A (Con A)-binding, 250-kilodalton glycoprotein characteristic of cerebellum. Extraction conditions for P400 protein were investigated, and complete solubilization of P400 protein from a submicrosomal fraction (P31 fraction) of mouse cerebellum was attained by the combination of 4% Zwittergent 3-14 and 4 M guanidinium chloride. The solubilized P400 protein was purified using Sepharose CL-4B and Con A-Sepharose chromatography. A monoclonal antibody (18A10) was prepared against P400 protein. Endo-beta-N-acetylglucosaminidase F digestion of P400 protein revealed that P400 protein has a small number of asparagine-linked oligosaccharide chains and that the epitope that is recognized by 18A10 monoclonal antibody is not on the asparagine-linked oligosaccharide portion. Tissue distribution of P400 protein was investigated by immunoblot analysis using 18A10 monoclonal antibody. P400 protein was abundant in the cerebellum, but a very small amount of P400 protein or related antigen was also detected in other parts of the nervous system and in nonneural tissues. Immunohistochemical studies indicated that P400 protein was distributed abundantly in the soma, the dendritic arborization, and the axon of the Purkinje cell. No immunoreaction was observed in the other types of cells.     

Reversibility of Cisternal Stack Formation During Hypoxic Hypoxia and Subsequent Reoxygenation in Cerebellar Purkinje Cells

Cisternal stacks are induced during hypoxia, which may be associated with intracellular Ca²⁺ regulation. Although neurons are divided internally in different compartments, little is known about regional differences in cisternal stack formation. We investigated the effects of hypoxic hypoxia and later reoxygenation on cisternal stack formation and other ultrastructual changes in the proximal dendrite, dendritic spine, and cell body of cerebellar Purkinje cells in rats. After brief hypoxic events, cisternal stacks appeared predominantly in the proximal dendrites and after longer hypoxic events in dendritic spines and cell body. Following reoxygenation, cisternal stacks disappeared first in the cell body, followed by the dendritic spines, then the proximal dendrites. These results showed that stack formation occurred at different degrees and time courses among the three regions, and the effect was reversible, which suggests that these compartments are differentially sensitive to hypoxia.     

Calcium Permeability of Non-N-Methyl-D-Aspartate Receptor Channels in Immature Cerebellar Purkinje Cells: Studies Using Fura-2 Microfluorometry

Abstract: Using fura-2 microfluorometry, I investigated the mechanism by which non-N-methyl-d -aspartate (NMDA) receptor agonists increase the cytosolic free calcium concentration ([Ca]_in) in single cerebellar Purkinje cells isolated from 3–10-day-old rats. Kainate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate dose-dependently increased the cytosolic free Na⁺ concentration, which was measured using sodium-binding benzofuran isophthalate microfluorometry, confirming the Na⁺ influx through ion channels linked to non-NMDA receptors. The [Ca²⁺] increases induced by relatively lower concentrations of agonists were entirely dependent on external Ca²⁺ and were reduced by removal of external Na⁺ or by addition of a Ca²⁺ channel blocker, D600. The results indicate that the non-NMDA agonist–induced [Ca]_in increase was due mainly to Ca²⁺ influx through voltage-dependent Ca²⁺ channels, which were activated by a massive Na⁺ influx. On the other hand, higher concentrations of agonists dose-dependently increased [Ca]_in under conditions in which activation of voltage-dependent Ca²⁺ channels were blocked by a combination of Na⁺ removal with D600. These [Ca]_in increases were Ca²⁺ dependent and little affected by adding a competitive NMDA antagonist. Non-NMDA agonists also stimulated influxes of Mn²⁺ and Co²⁺, both of which can be monitored by quenching fura-2 fluorescence under the same conditions. These results suggest that ion channels linked to non-NMDA receptors on immature Purkinje cells are permeable to Ca²⁺, Mn²⁺, and Co²⁺.     

Characterization of Microtubule-Associated Protein 2 from Mouse Brain and Its Localization in the Cerebellar Cortex

Microtubule-associated protein (MAP) 2 was purified from the microtubule fraction of mouse brain by heat treatment and BioGel A-5m gel filtration. The purified preparation showed a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis using both a gradient gel (3.75-12.5%) and a low-percentage gel (5%), a finding indicating that MAP2B was absent under the conditions used. Amino acid analysis revealed that mouse MAP2 was an acidic protein with an isoelectric point (pI 4.5) and amino acid composition similar to those of porcine brain MAP2. Immunoblot analysis indicated that the antigens that reacted with MAP2 antiserum were present in large quantities in mouse brain. However, we also found a weak reaction in various tissues other than brain, and the major antigens involved were recognized to be common molecular species with the same molecular mass, 162 and 170 kilodaltons. Using antiserum against mouse brain MAP2, the developmental localization patterns of MAP2 in the mouse cerebellar cortex were studied by immunohistochemistry. MAP2 was mainly localized in the neuronal cells throughout development, with the expression in Purkinje cell dendrites being especially remarkable in the growth of arborization from postnatal day 3 to day 20. At the mature stage, the reaction was strong in the dendritic tree but very weak in the proximal dendrites and cell bodies.     

Why Mouse Airway Submucosal Gland Serous Cells Do Not Secrete Fluid in Response to cAMP Stimulation

Airway submucosal glands are important sites of cystic fibrosis transmembrane conductance regulator (CFTR) chloride (Cl⁻) channel expression and fluid secretion in the airway. Whereas both mouse and human submucosal glands and their serous acinar cells express CFTR, human glands and serous cells secrete much more robustly than mouse cells/glands in response to cAMP-generating agonists such as forskolin and vasoactive intestinal peptide. In this study, we examined mouse and human serous acinar cells to explain this difference and reveal further insights into the mechanisms of serous cell secretion. We found that mouse serous cells possess a robust cAMP-activated CFTR-dependent Cl⁻ permeability, but they lack cAMP-activated calcium (Ca²⁺) signaling observed in human cells. Similar to human cells, basal K⁺ conductance is extremely small in mouse acinar cells. Lack of cAMP-activated Ca²⁺ signaling in mouse cells results in the absence of K⁺ conductances required for secretion. However, cAMP activates CFTR-dependent fluid secretion during low-level cholinergic stimulation that fails to activate secretion on its own. Robust CFTR-dependent fluid secretion was also observed when cAMP stimulation was combined with direct pharmacological activation of epithelial K⁺ channels with 1-ethyl-2-benzimidazolinone (EBIO). Our data suggest that mouse serous cells lack cAMP-mediated Ca²⁺ signaling to activate basolateral membrane K⁺ conductance, resulting in weak cAMP-driven serous cell fluid secretion, providing the likely explanation for reduced cAMP-driven secretion observed in mouse compared with human glands.