Intracellular pH (pHi) regulates several aspects of mammalian sperm function, although the transport mechanisms that control pHi in these cells are not understood. The pHi of mouse cauda epididymal sperm was determined from the fluorescence excitation ratio of 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein and calibrated with nigericin and elevated external [K+]. Two acid efflux mechanisms were identified following imposition of acid loads. One pathway has many anticipated characteristics of the somatic Na(+)-dependent Cl(-)-HCO3- exchanger, although sperm and somatic mechanisms can be distinguished by their ion selectivity and inhibitor sensitivity. Sperm may have an isoform of this exchange pathway with novel functional characteristics. The second acid-export pathway does not require extracellular anions or cations and is inhibited by arylaminobenzoates (flufenamic acid, diphenylamine-2-carboxylate). Mouse sperm also recover spontaneously from intracellular alkalinization. Recovery rates in N-methyl-D-glucamine+ Cl- or in 0.25 M sucrose are not significantly different from that in a complex culture medium. Thus, recovery from alkalinization does not utilize specific, ion-dependent transport mechanisms. Other widely distributed acid-efflux mechanisms, such as the Na(+)-H+ antiport pathway and the Na(+)-independent Cl(-)-HCO3- exchanger are not major regulators of mouse sperm pHi. Sperm capacitation results in pHi increases (from 6.54 +/- 0.08 to 6.73 +/- 0.09) that require a functional Na(+)-, Cl(-)-, and HCO3(-)-dependent acid-efflux pathway. Inhibition of this regulatory mechanism attenuates alkaline shifts in pHi during capacitation as well as the ability of sperm to produce a secretory response to zona pellucida agonists. These data suggest that one aspect of mouse sperm capacitation is the selective activation of one major pHi regulator.

Rat basophilic leukemia (RBL-2H3) cells predominantly express the type II receptor for inositol 1,4,5-trisphosphate (InsP 3 ), which operates as an InsP 3 -gated calcium channel. In these cells, cross-linking the high-affinity immunoglobulin E receptor (Fc⑀R1) leads to activation of phospholipase C ␥ isoforms via tyrosine kinase-and phosphatidylinositol 3-kinase-dependent pathways, release of InsP 3 -sensitive intracellular Ca 2ϩ stores, and a sustained phase of Ca 2ϩ influx. These events are accompanied by a redistribution of type II InsP 3 receptors within the endoplasmic reticulum and nuclear envelope, from a diffuse pattern with a few small aggregates in resting cells to large isolated clusters after antigen stimulation. Redistribution of type II InsP 3 receptors is also seen after treatment of RBL-2H3 cells with ionomycin or thapsigargin. InsP 3 receptor clustering occurs within 5-10 min of stimulus and persists for up to 1 h in the presence of antigen. Receptor clustering is independent of endoplasmic reticulum vesiculation, which occurs only at ionomycin concentrations Ͼ1 M, and maximal clustering responses are dependent on the presence of extracellular calcium. InsP 3 receptor aggregation may be a characteristic cellular response to Ca 2ϩ -mobilizing ligands, because similar results are seen after activation of phospholipase C-linked G-protein-coupled receptors; cholecystokinin causes type II receptor redistribution in rat pancreatoma AR4 -2J cells, and carbachol causes type III receptor redistribution in muscarinic receptor-expressing hamster lung fibroblast E36 M3R cells. Stimulation of these three cell types leads to a reduction in InsP 3 receptor levels only in AR4 -2J cells, indicating that receptor clustering does not correlate with receptor down-regulation. The calcium-dependent aggregation of InsP 3 receptors may contribute to the previously observed changes in affinity for InsP 3 in the presence of elevated Ca 2ϩ and/or may establish discrete regions within refilled stores with varying capacity to release Ca 2ϩ when a subsequent stimulus results in production of InsP 3 . INTRODUCTIONCross-linking the immunoglobulin E (IgE)-primed Fc⑀ receptor 1 (Fc⑀R1) of rat basophilic leukemia (RBL-2H3) cells leads to Lyn-mediated phosphorylation of immunoreceptor tyrosine activation motifs within the cytoplasmic tails of Fc⑀R1 ␤ and ␥ subunits, followed by recruitment and activation of the tyrosine kinase Syk (reviewed in Benhamou, 1997). This initial kinase activation results in stimulation of two isoforms of phospholipase C-␥, PLC␥1 and PLC␥2, and leads to elevated levels of inositol 1,4,5-trisphosphate (InsP 3 ) that are sustained over prolonged periods (Ͼ10 -15 min) of cross-linking (reviewed in Wilson et al., 1997). Previous evidence has shown that phosphatidylinositol 3-kinase supports the activation and phosphorylation of PLC␥1 and is required for maximal InsP 3 synthesis (Barker et al., 1995 (Fasolato, et al., 1993), although there is evidence that a second Ca 2ϩ influx pathway also participates in Ca...

Activation of certain phosphoinositidase-C-linked cell-surface receptors is known to cause an acceleration of the proteolysis of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptors and, thus, lead to Ins(1,4,5)P3-receptor down-regulation. In the current study we have sought to determine whether the ubiquitin/proteasome pathway is involved in this adaptive response. The data presented show (i) that activation of phosphoinositidase-C-linked receptors causes Ins(1,4,5)P3-receptor ubiquitination in a range of cell types (AR4-2J cells, INS-1 cells and rat cerebellar granule cells), (ii) that the Ins(1,4,5)P3-receptor down-regulation induced by activation of these receptors is blocked by proteasome inhibitors, (iii) that all known Ins(1,4,5)P3 receptors (types I, II and III) are substrates for ubiquitination, (iv) that ubiquitination occurs while Ins(1,4,5)P3 receptors are membrane-bound, (v) that Ins(1,4, 5)P3-receptor ubiquitination and down-regulation are stimulated only by those agonists that elevate Ins(1,4,5)P3 concentration persistently, and (vi) that a portion of cellular Ins(1,4,5)P3 receptors (those that are not type-I-receptor-associated) can be resistant to ubiquitination and degradation. In total these data indicate that the ubiquitin/proteasome pathway mediates Ins(1,4, 5)P3-receptor down-regulation and suggest that ubiquitination is stimulated by the binding of Ins(1,4,5)P3 to its receptor.

Despite its importance as a key parameter of child health and development, growth velocity is difficult to determine in real timebecause skeletal growth is slowand clinical tools to accurately detect very small increments of growth do not exist. We report discovery of a marker for skeletal growth in infants and children. The intact trimeric noncollagenous 1 (NC1) domain of type X collagen, the markerwe designated as CXMfor Collagen X Marker, is a degradation by-product of endochondral ossification that is released into the circulation in proportion to overall growth plate activity. Thismarker corresponds to the rate of linear bone growth at timeofmeasurement. Serumconcentrations of CXMplotted against age showa pattern similar to well-established height growth velocity curves and correlate with height growth velocity calculated from incremental height measurements in this study. The CXM marker is stable once collected and can be accurately assayed in serum, plasma, and dried blood spots. CXMtestingmay be useful for monitoring growth in the pediatric population, especially responses of infants and children with genetic and acquired growth disorders to interventions that target the underlying growth disturbances. The utility of CXM may potentially extend to managing other conditions such as fracture healing, scoliosis, arthritis, or cancer.