Platelets respond to various stimuli with rapid changes in shape followed by aggregation and secretion of their granule contents. Platelets lacking the α-subunit of the heterotrimeric G protein Gq do not aggregate and degranulate but still undergo shape change after activation through thromboxane-A2 (TXA2) or thrombin receptors. In contrast to thrombin, the TXA2 mimetic U46619 led to the selective activation of G12 and G13 in Gαq-deficient platelets indicating that these G proteins mediate TXA2 receptor-induced shape change. TXA2 receptor-mediated activation of G12/G13 resulted in tyrosine phosphorylation of pp72syk and stimulation of pp60c-src as well as in phosphorylation of myosin light chain (MLC) in Gαq-deficient platelets. Both MLC phosphorylation and shape change induced through G12/G13 in the absence of Gαq were inhibited by the C3 exoenzyme from Clostridium botulinum, by the Rho-kinase inhibitor Y-27632 and by cAMP-analogue Sp-5,6-DCl-cBIMPS. These data indicate that G12/G13 couple receptors to tyrosine kinases as well as to the Rho/Rho-kinase–mediated regulation of MLC phosphorylation. We provide evidence that G12/G13-mediated Rho/Rho-kinase–dependent regulation of MLC phosphorylation participates in receptor-induced platelet shape change.
The occurrence of the endogenous regulatory response to high rates of 2-deoxyglucose (2-DG) uptake, as previously described for C6 glioma cells during incubation with 2 mM 2-DG (Lange et al.: J. Cell. Physiol., 1989), was studied in 3T3-L1 preadipocytes and adipocytes, and the influence of insulin on this endogenous uptake regulation was examined. In contrast to 3T3-L1 preadipocytes, insulin-sensitive differentiated 3T3-L1 adipocytes displayed the time-dependent cyclic pattern of 2-DG uptake rates characteristic of the membrane-limited and endogenously regulated cellular state of hexose utilization. Although insulin induced a threefold stimulation of 2-DG tracer uptake in adipocytes, the hormone did not additionally stimulate the uptake rates or affect the periodic response: maximum and minimum levels of uptake remained unchanged. Scanning electron microscopy (SEM) revealed that the acquirement of the differentiated state is accompanied by a conspicuous transformation of the smooth surface of undifferentiated 3T3-L1 cells into a surface covered by numerous microvilli of uniform size and appearance. Treatment with insulin (10 mU/ml; 10 minutes) converted these microvilli into voluminous saccular membrane protrusions of the same type as had been formed during incubation of 3T3-L1 adipocytes with 2 mM 2-DG, and which have previously been shown to be involved in the endogenous uptake regulation of C6 glioma cells (Lange et al.: J. Cell. Physiol., 1989). These insulin-induced saccated membrane areas appeared to become integrated into the cell surface. Accordingly, insulin treatment caused a twofold increase of the intracellular distribution space of 3-O-methylglucose (3-OMG) in 3T3-L1 adipocytes. This insulin-induced increase of the 3-OMG distribution space exhibited the same time (t1/2 = 2-2.5 minutes) and dose dependence (EC50 = 20 nM) as the insulin-induced stimulation of 3-OMG transport. Glucose deprivation during the differentiation period inhibited the outgrowth of microvilli from the cell surface. Glucose starvation (18 hours at less than 0.5 mM) induced a conspicuous reduction of the length of microvilli on differentiated 3T3-L1 cells. In this state, the stalks of the microvilli are almost invisible and the enlarged spherical tips of the microvilli (with an average diameter of 370 nm compared to 230 nm of fed cells) appeared to protrude directly out of the cell surface. Starvation-induced shortening of microvilli was accompanied by a threefold increase of the basal 3-OMG transport rate and a greater than twofold increase of the intracellular 3-OMG distribution space as compared to fed cells (10 mM; 18 hours).(ABSTRACT TRUNCATED AT 400 WORDS)
Preceding studies have shown that the bulk of the ATP-dependent, inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ store of hamster insulinoma (HIT) cells is located in microvilli on the cell surface. Similar results were obtained with isolated rat hepatocytes. Moreover, in vesicles of microvillar origin, passive fluxes of Ca2+, ATP, and IP3 occur through cation and anion channels, respectively, suggesting that Ca2+ storage is due to ATP-dependent Ca2+ binding to an intravesicular component. Here we demonstrate that F-actin may be a possible candidate for this function. ATP-actin monomers bind Ca2+ with high affinity (Kd = 2-8 nM) to their divalent cation binding sites. Polymerization of actin monomers decreases the rate constant for divalent cation exchange at this binding site by more than 3 orders of magnitude rendering bound cations nearly unavailable. F-actin-bound Ca2+ can be released by depolymerization and dissociation from Ca(2+)-ADP-actin monomers (Kd = 375 nM). We now provide additional evidence for the possible involvement of actin in Ca2+ storage. (1) Preincubation of surface-derived Ca(2+)-storing vesicles from HIT cells with the F-actin stabilizer, phalloidin, strongly inhibited ATP-dependent Ca2+ uptake, reducing the IP3-sensitive Ca2+ pool by 70%. Phalloidin, when added after the loading process, affected neither the amount of stored Ca2+ nor IP3 action on the store. (2) F-actin polymerized in the presence of Mg2+ in nominally Ca(2+)-free buffer still contained about half of the high affinity sites occupied with Ca2+ (Mg/Ca-F-actin). (3) Using the fura-2 technique, we found that in the presence of ATP, Mg/Ca-F-actin incorporated free Ca2+ at a relatively low rate. Short pulses of ultrasound (3-10 s) strongly accelerated Ca2+ uptake, decreasing free Ca2+ from 500 nM to below 100 nM. (4) In the presence of physiological levels of Mg2+ (0.5 mM), sonication liberated large amounts of Ca2+ from Mg/Ca-F-actin. (5) Ca-F-actin released bound Ca2+ at a very slow rate. Short ultrasonic pulses rapidly elevated free Ca2+ from about 50 nM up to 500 nM. (6) Small amounts of profilin, an actin-binding protein, released Ca2+ both from Ca- and Mg/Ca-F-actin and also inhibited uptake of Ca2+ into Mg/Ca-F-actin. (7) Phalloidin completely inhibited Ca-uptake into Mg/Ca-F-actin even during ultrasonic treatment. These findings suggest that Ca2+ storage may occur by addition of Ca-ATP-actin monomers to reactive ends of the polymer and emptying of this store by profilin-stimulated release of Ca-ADP-actin. Thus, receptor-operated Ca2+ signaling, initiated by phospholipase C activation, may proceed via the well-known phosphatidylinositol phosphate-regulated profilin/gelsolin pathway of actin reorganization/depolymerization. The importance of the proposed microvillar Ca2+ signaling system for living cells remains to be established.
Electron microscopic and biochemical techniques were used to study the cellular localization of the ATP‐dependent, IP3‐sensitive, Ca2+ store in the glucose‐ and phosphatidylinositol(PI) agonist‐sensitive hamster insulinoma cell line HIT‐T15. Scanning electron microscopy revealed conspicuous shape changes of the microvilli following stimulation of these cells with bombesin or thapsigargin. These changes closely resemble those previously shown to accompany stimulation of hexose transport in adipocytes with insulin [J. Cell. Physiol. 142 (1990) 1‐14]. Using a hydrodynamic shearing technique for the isolation of microvilli, two cell surface‐derived vesicle fractions were prepared containing 80% of the total cellular Ca2+‐storing activity. In contrast, subcellular fractionation using normal homogenization with a glass/teflon homogenizer yielded the well‐known distribution of the Ca2+‐storing activity which is then predominantly recovered within the microsomal fraction. The surface‐derived vesicle fraction was clearly distinguished from the microsomal fraction by its high content of Na+/K+‐ATPase and an immunoreactive fragment of the GluT‐1 glucose transporter isoform which both are not detectable in the microsomal fraction isolated from homogenates from sheared cells. The Ca2+ uptake properties of the cell surface‐derived vesicle fractions including the vanadate, A23187, and thapsigargin sensitivity were found to be identical with those described for the microsomal Ca2+ stores of various cell types. Inositol 1,4,5‐trisphosphate (IP3) at 1 μM induced a maximal release of 35–40% of the stored Ca2+ from these vesicles.
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