Influence of PMA and a low extracellular Ca2+ concentration on the development of the Na(+)-dependent hexose carrier in LLC-PK1 cells.

We have analyzed the development of Na(+)-dependent hexose transport during differentiation and during polarization of LLC-PK1, an established cell line with characteristics of the proximal tubule. When cell-cell contact was disturbed by a low extracellular Ca2+ concentration or by a phorbol myristate acetate (PMA) treatment, the development of Na(+)-dependent hexose transport was completely inhibited. The effect of PMA on the development of hexose transport could be uncoupled from its effect on the tight junctions. The PMA concentration needed for the latter effect was approx. 10-fold higher than for the former. As the primary cause of the PMA effect, an influence on the cytoskeleton is suggested. In contrast to PMA, the concentration dependence of both phenomena on the extracellular Ca2+ concentration was almost the same. Moreover, the incorporation of hexose carriers in the plasma membrane could be induced by changing the extracellular CA2+ concentration from low to normal. We conclude that there is a relation between the formation of tight junctions and the development of the Na(+)-dependent hexose carrier, possibly because Ca(2+)-dependent cell adhesion molecules play a role in both phenomena. However, a direct relation between Ca(2+)-dependent elements of the tight junctions and the insertion of the hexose carrier can not be excluded. The Ca(2+)-dependent development seems to be a common characteristic of apical membrane proteins in contrast to the development of the basolateral membrane protein, (Na(+)+K+)-ATPase.     

Mechanism of Ca2+ inhibition of inositol 1,4,5-trisphosphate (InsP3) binding to the cerebellar InsP3 receptor.

Ca2+ efficiently inhibits binding of inositol 1,4,5-trisphosphate (InsP3) to the InsP3 receptor in cerebellar membranes but not to the purified receptor. We have now investigated the mechanism of action by which Ca2+ inhibits InsP3 binding. Our results suggest that Ca2+ does not cause the stable association of a Ca(2+)-binding protein with the receptor. Instead, Ca2+ leads to the production of a soluble, heat-stable, low molecular weight substance from cerebellar membranes that competes with InsP3 for binding. This inhibitory substance probably represents endogenously generated InsP3 as judged by the fact that it co-purifies with InsP3 on anion-exchange chromatography, competes with [3H]InsP3 binding in a pattern similar to unlabeled InsP3, and is in itself capable of releasing 45Ca2+ from permeabilized cells. A potent Ca(2+)-activated phospholipase C activity producing InsP3 was found in cerebellar microsomes that exhibited a Ca2+ dependence identical to the Ca(2+)-dependent inhibition of InsP3 binding. Together these results suggest that the Ca(2+)-dependent inhibition of InsP3 binding to the cerebellar receptor is due to activation of a Ca(2+)-sensitive phospholipase C enriched in cerebellum. Nevertheless, Ca2+ probably also modulates the InsP3 receptor function by a direct interaction with the receptor that does not affect InsP3 binding but regulates InsP3-dependent channel gating.     

Effect of protein kinase C activation and Ca2+ mobilization on hexose transport in Swiss 3T3 cells

Down-modulation of Ca²⁺-activated, phospholipid-dependent protein kinase (protein binase C), which was accomplished by pretreatment with phorbol-12,13-dibutyrate for 24 h, resulted in the loss of a phorbol ester-induced stimulation of hexose transport activity in Swiss 3T3 cells. In these cells, however, platelet-derived growth factor as well as Ca²⁺ ionophore A23187 were still able to induce stimulation of hexose transport activity accompanied by the elevation of intracellular free Ca²⁺ concentration. Since chelation of extracellular Ca²⁺ inhibited this stimulation, inflow of extracellular Ca²⁺ into cytoplasm seemed to be esential for the stimulatory effect of platelet-derived growth factor and A23187 on hexose transport. Epidermal growth factor and insulin also stimulated hexose transport activity regardless of the absence of protein kinase C. However, in the case of epidermal growth factor, intracellular Ca²⁺, but not extracellular Ca²⁺, was found to be necessary for the stimulation. On the other hand, insulin stimulated the hexose transport independent of both intra- and extracellular Ca²⁺.     

Role of calcium in serum-stimulation of hexose transport in muscle cells

Serum stimulates glucose uptake into several cells in culture. In intact muscle, an increase in cytosolic free Ca2+ has been proposed to mediate the activation of glucose uptake by hormones and other stimuli [Cell Calcium (1980) 1, 311-325]. We report that hexose (2-deoxy-D-glucose) uptake into L6 muscle cells in culture is enhanced several-fold by fetal calf serum. The increase in uptake is due to stimulation of transmembrane transport, since serum also stimulated uptake of the non-metabolizable hexose 3-O-methyl-D-glucose. The role of Ca2+ in this stimulation was assessed: (i) stimulation of transport by serum was independent of the presence of extracellular Ca2+ during the incubation with serum; (ii) the intracellular levels of free Ca2+, measured by the fluorescence of the novel Ca-indicator quin-2, were identical in serum-stimulated and control cells. It is concluded that hexose transport can increase in muscle cells without concomitant changes in cytoplasmic free Ca2+.     

Sarcolemmal glucose transport in Ca2+-tolerant myocytes from adult rat heart. Calcium dependence of insulin action

Cardiac myocytes were isolated from adult rat ventricles by a method which preserves their functional integrity, including long survival in physiological concentrations of Ca2+. Sarcolemmal glucose transport was assessed by measuring linear initial uptake rates of the nonmetabolized glucose analog 3-O-methyl-D-glucose. Transport was saturable and showed competition by D-glucose and other features of chemical and stereo-selectivity. Transport was stimulated by insulin in a dose-dependent manner, resulting in an almost 5-fold increase in Vmax, with little change in Km. Stimulation of 3-methylglucose transport by insulin was largely Ca2+-dependent. Omission of Ca2+ from the incubation medium caused a minor rise in basal 3-methylglucose uptake but the insulin-stimulated rise in Vmax was only 30%. The Ca2+ antagonist D600 also antagonized stimulation of hexose transport by insulin. In all the above respects, 3-methylglucose transport in myocytes is identical to that in intact heart muscle. In addition, the decrease in insulin response by Ca2+ omission was partially reversed by subsequent return to a Ca2+-containing medium. ATP levels remained stable in the absence of Ca2+, showing that the Ca2+ dependence did not reflect nonspecific cell damage.     

IRBIT, inositol 1,4,5-triphosphate (IP3) receptor-binding protein released with IP3, binds Na+/H+ exchanger NHE3 and activates NHE3 activity in response to calcium

Calcium (Ca2+) is a highly versatile second messenger that regulates various cellular processes. Previous studies showed that elevation of intracellular Ca2+ regulates the activity of Na+/H+ exchanger 3 (NHE3). However, the effect of Ca2+-dependent signaling on NHE3 activity varies depending on cell types. In this study, we report the identification of IP3 receptor-binding protein released with IP3 (IRBIT) as a NHE3 interacting protein and its role in regulation of NHE3 activity. IRBIT bound to the carboxyl-terminal domain of NHE3, which is necessary for acute regulation of NHE3. Ectopic expression of IRBIT resulted in Ca2+-dependent activation of NHE3 activity, whereas silencing of endogenous IRBIT resulted in inhibition of NHE3 activity. Ca2+-dependent stimulation of NHE3 activity was dependent on the binding of IRBIT to NHE3. Previously Ca2+-dependent inhibition of NHE3 was demonstrated in the presence of NHERF2. Co-expression of IRBIT was able to reverse the NHERF2-dependent inhibition of NHE3. We also showed that IRBIT-dependent activation of NHE3 involves exocytic trafficking of NHE3 to the plasma membrane and this activation was blocked by inhibition of calmodulin (CaM) or CaM-dependent kinase II. These results suggest that the overall effect of Ca2+ on NHE3 activity is balanced by IRBIT-dependent activation and NHERF2-dependent inhibition.     

Ca2+-dependent translocation of hexose carrier in mouse fibroblast Swiss 3T3 cells

Ca2+-induced translocation of hexose carriers from microsomal membrane to plasma membrane was demonstrated in saponin-permeabilized Swiss 3T3 cells by a specific D-glucose-inhibitable cytochalasin B-binding assay. The number of hexose carriers in the plasma membrane and the hexose transport activity in intact cells were also compared. The incubation of permeabilized cells with 10 microM Ca2+ at 37 degrees C rapidly increased the number of D-glucose-inhibitable cytochalasin B-binding sites in the plasma membrane from 13 to 40 pmol/mg protein and concomitantly decreased that in the microsomal membrane from 66 to 36 pmol/mg protein, each with a half-time of approx. 2 min. Furthermore, when Ca2+-stimulated cells were exposed to 50 microM EGTA, the effect of Ca2+ on the translocation of D-glucose-inhibitable cytochalasin B-binding sites was reversed with a half-time of approx. 5 min. The concentration of Ca2+ required for the half-maximal effect was approx 500 nM. The magnitude of the stimulatory effect of D-glucose-inhibitable cytochalasin B-binding sites in the plasma membrane closely correlated with the magnitude of stimulatory action of Ca2+ on 3-O-methylglucose transport in the intact cells. These results suggest that Ca2+ regulates the activity of hexose transport across the plasma membrane through a rapid and reversible translocation of hexose carrier between microsomal and plasma membranes of mouse fibroblast Swiss 3T3 cells.     

A two sodium ion/D-glucose symport mechanism: membrane potential effects on phlorizin binding

Apical membrane vesicles isolated from a continuous renal cell line, LLC-PK1, catalyze electrogenic Na+-stimulated hexose transport and Na+-dependent binding of 3H-labeled 1-[2-(beta-D-glucopyranosyloxy)-4, 6-dihydroxyphenyl]-3-(4-hydroxyphenyl)-1-propanone [( 3H]phlorizin), a competitive ligand of this transport system. Phlorizin was not itself transported across the membrane and thus can serve as a probe of the binding step. The stoichiometry of Na+-dependent phlorizin binding in vesicles was 1:1, whereas Na+/hexose cotransport in vesicles exhibited a 2:1 stoichiometry. Na+ increased the affinity of phlorizin binding without affecting the total number of binding sites. An increased number of Na+-dependent phlorizin binding sites was observed under conditions of interior-negative membrane potential. These results are consistent with a model of the Na+/glucose cotransport cycle in which the unloaded transporter is negatively charged and its orientation influenced by membrane potential. Glucose and one sodium ion interact with the transporter, resulting in an uncharged complex. Binding of a second sodium ion triggers translocation of glucose and both sodium ions via formation of a loaded carrier complex bearing a single positive charge.     

Dissociation of 5' AMP-activated protein kinase activation and glucose uptake stimulation by mitochondrial uncoupling and hyperosmolar stress: differential sensitivities to intracellular Ca2+ and protein kinase C inhibition

2,4-dinitrophenol (DNP) compromises ATP production within the cell by disrupting the mitochondrial electron transport chain. The resulting loss of ATP leads to an increase in glucose uptake for anaerobic generation of ATP. In L6 skeletal muscle cells, DNP increases the rate of glucose uptake by twofold. We previously showed that DNP increases cell surface levels of glucose transporter 4 (GLUT4) and hexose uptake via a Ca2+-sensitive and conventional protein kinase C (cPKC)-dependent mechanism. Recently, 5' AMP-activated protein kinase (AMPK) has been proposed to mediate the stimulation of glucose uptake by energy stressors such as exercise and hypoxia. Changes in Ca2+ and cPKC have also been invoked in the stimulation of glucose uptake by exercise and hypoxia. Here we examine whether changes in cytosolic Ca2+ or cPKC lead to activation of AMPK. We show that treatment of L6 cells with DNP (0.5 mM) or hyperosmolar stress (mannitol, 0.6 M) increased AMPK activity by 3.5-fold. AMPK activation peaked by 10-15 min prior to maximal stimulation of glucose uptake. Intracellular Ca2+ chelation and cPKC inhibition prior to treatment with DNP and hyperosmolarity significantly reduced cell surface GLUT4 levels and hexose uptake but had no effect on AMPK activation. These results illustrate a break in the relationship between AMPK activation and glucose uptake in skeletal muscle cells. Activation of AMPK does not suffice to stimulate glucose uptake in response to DNP and hyperosmolarity.     

Absence of active protein kinase C in ram spermatozoa

At fertilization, mammalian spermatozoa undergo a Ca2+-dependent exocytotic event known as the acrosome reaction. As protein kinase C (PKc) has been implicated in exocytosis in some other cell systems, we have searched for PKc in ram spermatozoa. We have found that: (a) no changes in protein phosphorylation pattern could be induced in the intact cells by phorbol dibutyrate (PDBu), a compound which binds to and stimulates PKc; (b) no changes in protein phosphorylation pattern could be detected during the course of the Ca2+/ionophore-induced acrosome reaction (when greater than 95% of the cells underwent exocytosis); (c) there was no effect of PDBu on the exocytotic response to various Ca2+ and ionophore levels; (d) no specific PDBu binding could be detected in the cells (this binding is considered to be indicative of the presence of active PKc). We conclude that potentially active PKc is not present in ram spermatozoa.     

Characterization of a membrane protein from brain mediating the inhibition of inositol 1,4,5-trisphosphate receptor binding by calcium.

Inositol 1,4,5-trisphosphate (InsP3) is a component of the phosphoinositide second-messenger system which mobilizes Ca2+ from intracellular stores. Recently, an InsP3 receptor binding protein from rat cerebellar membranes was solubilized and purified to homogeneity. The potent inhibition by Ca2+ of [3H]InsP3 binding to the InsP3 receptor in cellular membranes is not apparent in the purified receptor. The Ca2+-dependent inhibition of [3H]InsP3 binding in the crude homogenate (concn. giving 50% inhibition = 300 nM) can be restored by addition of solubilized cerebellar membranes to the purified receptor. In the present study, we further characterize the protein in solubilized membranes which confers Ca2+-sensitivity to the receptor, and which we term 'calmedin'. Calmedin appears to be a neutral membrane protein with an estimated Mr of 300,000 by gel filtration in the presence of Triton X-100. Calmedin confers a Ca2+-sensitivity to InsP3 receptor binding, which can be completely reversed by 10 min incubation with EDTA and therefore does not represent Ca2+-dependent proteinase action. Calmedin effects on the purified InsP3 receptor depend on Ca2+ binding to the calmedin, although Ca2+ also binds directly to the InsP3 receptor. The regional distribution of calmedin differs from that of the InsP3 receptor in the brain, suggesting that it also mediates other Ca2+-dependent functions. Calmedin activity in peripheral tissues is much lower than in brain.     

Auranofin increases the affinity of phorbol dibutyrate receptors in chronic lymphocytic leukemia cells (B cells)

Previous studies have shown that auranofin (AF), a lipophilic gold I complex, modulates metabolic events in leukocytes stimulated by phorbol esters, whose major cellular binding site is now known to be the Ca++/phospholipid-dependent protein kinase (protein kinase C). In these experiments we have investigated the effect of AF on the binding of phorbol dibutyrate (PDBu) to human chronic lymphocytic leukemia (CLL) B cells. AF enhanced binding of PDBu to its receptor in CLL cells by a) causing an increase in the affinity of PDBu receptors from Kd 20.3 nM to 7.3 nM, and b) enhancing translocation of PDBu receptors to the cell membrane. The increase in PDBu binding induced by AF in whole cells was only partially reversible by EGTA or the intracellular Ca++ antagonist TMB-8. Studies performed with quin-2-labeled cells showed that 100 microM AF caused a mean (+/- SD) rise in cytosolic Ca++ levels from 0.41 (0.12) to 0.85 (0.33) (n = 5). Thus the mechanism by which AF increases binding of PDBu to its receptor appears to be partially dependent on Ca++. These effects of AF occurred at cellular levels achieved in mononuclear cells during chrysotherapy of patients with rheumatoid arthritis.     

Cytoplasmic Ca2+ during differentiation of 3T3-L1 adipocytes. Effect of insulin and relation to glucose transport

The cytoplasmic concentration of ionized Ca2+ [( Ca2+]i) was determined in 3T3-L1 cells during their differentiation from fibroblasts to adipocytes, suspended and loaded with the fluorescent Ca2+ indicators quin2 or indo-1. In undifferentiated fibroblasts, as well as in differentiated adipocytes up to day 9, [Ca2+]i was steady around 170 nM, and it increased significantly only in old adipocytes (day 12). During differentiation, stimulation of glucose uptake by insulin increased from a few percent to severalfold. Stimulation of uptake was already apparent after 10 min of addition of the hormone, and 10 nM insulin produced maximal stimulation in 30 min. Insulin (10(-6) M) added to quin2- or indo-1-loaded, suspended adipocytes had no detectable effect on [Ca2+]i for at least 10 min. In contrast, addition of the general anesthetic halothane increased [Ca2+]i from 172 to 251 nM in 3 min. In EGTA solution, the Ca2+ ionophore ionomycin elicited release of Ca2+ from intracellular stores that resulted in a transient increase in [Ca2+]i. A smaller but measurable Ca2+ release from intracellular stores (increasing [Ca2+]i by 20 nM) resulted upon addition of 20 micrograms/ml phosphatidic acid. In contrast, insulin did not produce any detectable release of Ca2+ from intracellular stores. Incubation of 3T3-L1 adipocytes with insulin in the presence of EGTA (the latter in excess over the Ca2+ concentration of the medium) did not prevent the stimulation of hexose uptake by the hormone, indicating that extracellular Ca2+ does not play a role in the insulin response. Furthermore, incubation of cells with quin2/AM in EGTA medium during exposure to insulin did not prevent stimulation of hexose uptake. Under these conditions it is demonstrated that intracellular quin2 suffices to chelate cytoplasmic Ca2+ even if releasable Ca2+ from intracellular stores were to pour into the cytoplasm. Thus, quin2 effectively lowers [Ca2+]i without impairing insulin action. It is concluded that insulin does not produce changes in [Ca2+]i and that chelating intracellular Ca2+ does not prevent stimulation of hexose uptake by insulin. These results suggest that it is unlikely that changes in [Ca2+]i may play a role in the transduction of information in insulin stimulation of glucose uptake in 3T3-L1 adipocytes.     

Regulation of 3-O-methyl-D-glucose uptake in isolated bovine adrenal chromaffin cells

We showed earlier that insulin stimulated sugar transport in adrenal chromaffin cells (Bigornia, L. and Bihler, I. Biochim. Biophys. Acta 885, 335-344). Transport regulation and its Ca2+ -dependence was further investigated in isolated bovine adrenal chromaffin cells, serving as a model of a homogeneous neuronal cell population. Uptake of the nonmetabolizable glucose analogue, 3-O-methyl-D-glucose was stimulated by hyperosmolar medium, and this effect was abolished in the absence of external Ca2+, or depressed in the presence of La3+ or the slow Ca2+ channel blocker methoxyverapamil. Basal transport was also stimulated by factors (acetylcholine, carbamylcholine, low-Na+ medium), which cause Ca2+ -dependent catecholamine release, and these effects were abolished in Ca2+ -free medium. In addition insulin, acetylcholine, hyperosmolar and low-Na+ medium significantly increased 45Ca uptake. Thus, glucose transport in adrenal chromaffin cells was stimulated by insulin and hyperosmolarity in a Ca2+ -dependent manner, as in muscle. Sensitivity to secretory stimuli, a regulatory feature perhaps characteristic of this cell type, was also demonstrated. In contrast to muscle, sugar transport was not affected by Na+ -pump inhibition, metabolic inhibitors or the Na+ ionophore monensin, suggesting that Ca2+ influx by Na+/Ca2+ exchange does not play a significant role in the activation of sugar transport in chromaffin cells.     

Functional specialization of calreticulin domains

Calreticulin is a Ca2+-binding chaperone in the endoplasmic reticulum (ER), and calreticulin gene knockout is embryonic lethal. Here, we used calreticulin-deficient mouse embryonic fibroblasts to examine the function of calreticulin as a regulator of Ca2+ homeostasis. In cells without calreticulin, the ER has a lower capacity for Ca2+ storage, although the free ER luminal Ca2+ concentration is unchanged. Calreticulin-deficient cells show inhibited Ca2+ release in response to bradykinin, yet they release Ca2+ upon direct activation with the inositol 1,4,5-trisphosphate (InsP3). These cells fail to produce a measurable level of InsP3 upon stimulation with bradykinin, likely because the binding of bradykinin to its cell surface receptor is impaired. Bradykinin binding and bradykinin-induced Ca2+ release are both restored by expression of full-length calreticulin and the N + P domain of the protein. Expression of the P + C domain of calreticulin does not affect bradykinin-induced Ca2+ release but restores the ER Ca2+ storage capacity. Our results indicate that calreticulin may play a role in folding of the bradykinin receptor, which affects its ability to initiate InsP3-dependent Ca2+ release in calreticulin-deficient cells. We concluded that the C domain of calreticulin plays a role in Ca2+ storage and that the N domain may participate in its chaperone functions.     

Bradykinin transiently activates protein kinase C in Swiss 3T3 cells. Distinction from activation by bombesin and vasopressin

The results presented here demonstrate that bradykinin, acting through a B2 subtype receptor, induces a unique pattern of early signals in quiescent Swiss 3T3 cells. Bradykinin caused a rapid mobilization of calcium from internal stores, as judged by measurements of intracellular Ca2+ concentration in fura-2-loaded cells and by 45Ca2+ efflux from radiolabeled cells. Analysis of phosphoproteins from 32P-labeled Swiss 3T3 cells by one- and two-dimensional gel electrophoresis revealed that bradykinin stimulated transient phosphorylation of an acidic cellular protein migrating with an apparent Mr = 80,000 (termed 80K), identified as a major and specific substrate of protein kinase C. Down-regulation of protein kinase C by pretreatment with phorbol 12,13-dibutyrate (PDBu) completely abolished the increase in 80K phosphorylation. In contrast to the sustained effect induced by bombesin, vasopressin, or PDBu, the stimulation of 80K phosphorylation by bradykinin reached a maximum after 1 min of incubation, and then it rapidly decreased to almost basal levels. Furthermore, bradykinin did not induce protein kinase C-mediated events such as inhibition of 125I-epidermal growth factor binding or enhancement of cAMP accumulation. Bombesin and vasopressin elicited both responses in parallel cultures. Bradykinin induced rapid accumulation of total inositol phosphates in cells labeled with myo-[3H]inositol. In contrast to bombesin and vasopressin which stimulated a linear increase in inositol phosphate accumulation over a 10-min period, the effect of bradykinin reached a plateau after 2.5 min of incubation with no further increase up to 10 min. The results demonstrate that the early signaling events triggered by bradykinin can be distinguished from those elicited by bombesin and vasopressin in Swiss 3T3 cells.     

Phosphorylation of brush border myosin I by protein kinase C is regulated by Ca(2+)-stimulated binding of myosin I to phosphatidylserine concerted with calmodulin dissociation.

Brush border myosin I from chicken intestine is phosphorylated in vitro by chicken intestinal epithelial cell protein kinase C. Phosphorylation on serine and threonine to a maximum of 0.93 mol of P/mol of myosin I occurs within an approximately 20 kDa region at the end of the COOH-terminal tail of the 119-kDa heavy chain. The effects of Ca2+ on myosin I phosphorylation by protein kinase C are complex, with up to 4-fold stimulation occurring at 0.5-3 microM Ca2+, and up to 80% inhibition occurring at 3-320 microM Ca2+. Phosphorylation required that brush border myosin I be in its phosphatidylserine vesicle-bound state. Previously unknown Ca2+ stimulation of brush border myosin I binding to phosphatidylserine vesicles was found to coincide with Ca2+ stimulation of phosphorylation. A myosin I proteolytic fragment lacking approximately 20 kDa of its tail retained Ca(2+)-stimulated binding, but showed reduced Ca(2+)-independent binding. Ca(2+)-dependent phosphatidylserine binding is apparently due to the concomitant phosphatidylserine-promoted, Ca(2+)-induced dissociation of up to three of the four calmodulin light chains from myosin I. Four highly basic putative calmodulin-binding sites in the Ca(2+)-dependent phosphatidylserine binding region of the heavy chain were identified based on the similarity in their sequence to the calmodulin- and phosphatidylserine-binding site of neuromodulin. Calmodulin dissociation is now shown to occur in the low micromolar Ca2+ concentration range and may regulate the association of brush border myosin I with membranes and its phosphorylation by protein kinase C.     

1,25-Dihydroxyvitamin D3 regulation of phorbol ester receptors in HL-60 leukemia cells

In this study the relationship between cell binding of phorbol 12,13-dibutyrate (PDBu) and induction of differentiation by 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) was examined. Binding of [3H]PDBu increased within 12 h of 1,25-(OH)2D3 treatment, and a 60-130% increase in [3H]PDBu receptor levels was observed within 24 h. By 48 h, however, [3H]PDBu binding was not different from control. Scatchard analysis of [3H]PDBu binding showed no statistical differences in Kd value (Kd approximately equal to 30 nM) between 1,25-(OH)2D3-treated and control cells 22 h post-treatment; however, a 2-fold increase in Bmax was observed in treated (338 +/- 24 pmol/10(9) cells) compared to control cultures (170 +/- 14 pmol/10(9) cells). Stimulation of [3H]PDBu binding was dependent on 1,25-(OH)2D3 concentrations over a range of 1-100 nM. Homogenates from 1,25-(OH)2D3-treated HL-60 cells also demonstrated an increase (70%) in [3H]PDBu binding to the Ca2+/phospholipid-dependent enzyme protein kinase C as assessed by incubation of cell homogenates with [3H]PDBu in the presence of saturating phosphatidylserine and calcium concentrations. This suggests that the increase in [3H]PDBu binding cannot be entirely explained by modulation of the latter two agents. Cycloheximide (5 microM), an inhibitor of protein synthesis, ablated the 1,25-(OH)2D3-stimulated increase in [3H]PDBu binding to intact HL-60 cells. These data demonstrate that an increase in [3H]PDBu binding occurs early in the course of 1,25-(OH)2D3-induced differentiation, results from an increased number of [3H]PDBu-binding site, and is dependent on protein synthesis.     

Two modes of regulation of the phospholipase C-linked substance-P receptor in rat parotid acinar cells.

In rat parotid acinar cells prelabelled with [3H]inositol, substance P (100 nM) induced the formation of [3H]inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. Ins(1,4,5)P3 reached a maximum 7 s after substance P stimulation, and thereafter decreased and reached a stable value at 60 s. When the cells were exposed to substance P for 10, 30, 60, or 300 s, washed, and re-exposed to this peptide, the formation of [3H]inositol trisphosphate (InsP3) was attenuated in a time-dependent manner. In the cells pretreated as described above, the number of [3H]substance-P-binding sites (Bmax) was also decreased. Possible role(s) of Ca2+ and protein kinase (protein kinase C) control mechanisms in regulating substance P responses were investigated. Desensitization of substance P-induced InsP3 was not affected by the Ca2+ ionophore ionomycin, nor was it dependent on Ca2+ mobilization. On the other hand, in the presence of 4 beta-phorbol 12,13-dibutyrate (PDBu) and 12-O-tetradecanoyl-4 beta-phorbol 13-acetate, known activators of protein kinase C, substance P-induced InsP3 formation was inhibited. However, PDBu had no effect on [3H]substance P binding, whether present during the assay or when cells were pretreated. The persistent desensitization of InsP3 formation induced by substance P was not affected by PDBu. These results suggest that the persistent desensitization of InsP3 formation induced by substance P is a homologous process involving down-regulation of the substance P receptor; the mechanism does not appear to involve, or to be affected by, the Ca2+ or protein kinase C signalling systems. Protein kinase C activation can, however, inhibit substance P-induced InsP3 formation, which may indicate the presence of a negative-feedback control on the substance P pathway.