The adenylate cyclase-coupled vasopressin V2-receptor is highly laterally mobile in membranes of LLC-PK1 renal epithelial cells at physiological temperature.

The lateral mobility of membrane-associated hormone receptors has been proposed to play an important role in signal transduction. Direct measurements, however, have shown that the receptors for insulin, epidermal growth factor and beta-adrenergic antagonists exhibit low mobility at physiological temperature. The present study, which represents the first report of lateral mobility of a polypeptide hormone receptor coupled to adenylate cyclase, yielded quite different results. The lateral mobility of the vasopressin renal-type (V2)-receptor was measured in the basal plasma membrane of cells of the LLC-PK1 porcine epithelial line, using the technique of fluorescence microphotolysis (photobleaching) and a rhodamine-labelled analogue of vasopressin. The analogue, 1-deamino[8-lysine(N6-tetramethylrhodamylaminothiocarbonyl)] vasopressin (TR-LVP) was synthesized and shown to have binding properties and biological activities very similar to those of Arg8-vasopressin (AVP). TR-LVP could be used to label specifically the V2-receptor of living LLC-PK1 cells, whereby LLC-PK1 cells incubated with TR-LVP in the presence of a 100-fold excess of AVP, or cells from the LLC-PK1 V2-receptor-deficient line M18 incubated with TR-LVP could be used as controls for non-specific binding. Using optical sectioning, specific receptor mobility could be measured both in the absence and presence of free TR-LVP. The V2-receptor was found to be largely mobile at 37 degrees C: the mobile fraction (f) was approximately 0.9, and the apparent lateral diffusion coefficient (D) approximately 3.0 X 10(-10) cm2/s. V2-receptor mobility greatly decreased with decreasing temperature: at 10 degrees C f was reduced to approximately 0.1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of arachidonic acid release by vasopressin in A7r5 vascular smooth muscle cells mediated by Ca2+-stimulated phospholipase A2

Arachidonic acid (AA) regulates many aspects of vascular smooth muscle behaviour, but the mechanisms linking receptors to AA release are unclear. In A7r5 vascular smooth muscle cells pre-labelled with (3)H-AA, vasopressin caused a concentration-dependent stimulation of 3H-AA release that required phospholipase C and an increase in cytosolic [Ca2+]. Ca2+ release from intracellular stores and Ca2+ entry via L-type channels or the capacitative Ca2+ entry pathway were each effective to varying degrees. Selective inhibitors of PLA2 inhibited the 3H-AA release evoked by vasopressin, though not the underlying Ca2+ signals, and established that cPLA2 mediates the release of AA. We conclude that in A7r5 cells vasopressin stimulates AA release via a Ca2+-dependent activation of cPLA2.     

Microtubule-dependent PKC-alpha localization in A7r5 smooth muscle cells

Using laser scanning confocal, fluorescence resonance energy transfer (FRET), and atomic force (AFM) microscopy, we investigated association of protein kinase C (PKC)- with microtubules during stimulus-induced relocalization in A7r5 smooth muscle cells. Confocal microscopy with standard immunostaining techniques confirmed earlier observations that colchicine disruption of microtubules blocked PKC- localization in the perinuclear region of the cell caused by phorbol 12,13-dibutyrate (PDBu; 10^–6M). Dual immunostaining suggested colocalization of PKC- and -tubulin in both unstimulated and PDBu-treated cells. This finding was verified by FRET microscopy, which indicated that association of PKC- was heterogeneous in distribution and confined primarily to microtubules in the perinuclear region. FRET analysis further showed that association between the molecules was not lost during colchicine-induced dissolution of microtubules, suggesting formation of tubulin-PKC- complexes in the cytosol. Confocal imaging indicated that perinuclear microtubular structure was more highly sensitive to colchicine dissolution than other regions of the cell. Topographic imaging of fixed cells by AFM indicated a well-defined elevated structure surrounding the nucleus that was absent in colchicine-treated cells. It was calculated that the volume of the nuclear sleevelike structure of microtubules increased approximately fivefold in PDBu-treated cells, suggesting a probable increase in microtubular mass. In light of PKC- localization, increased colchicine sensitivity, and their volume change in stimulated cells, the results suggest that perinuclear microtubules form a specialized structure that may be more dynamically robust than in other regions of the cell. PKC- could contribute to this dynamic activity. Alternatively, perinuclear microtubules could act as a scaffold for regulatory molecule interaction at the cell center. cytoskeleton; protein kinase C-; translocation     

Isolation of tocopherol-binding proteins from the cytosol of smooth muscle A7r5 cells.

Cultured smooth muscle A7r5 cells were able to take up alpha-tocopherol (32 +/- 1.2 nmol/mg protein) the largest part of which (60%) was present in the cytosolic fraction. Using a tocopherol-based affinity chromatography and alpha-, beta-, gamma-, and delta-tocopherols as eluants, three polypeptides of molecular masses 81, 58 and 31 kDa were eluted. This preparation had alpha-[3H]tocopherol binding capability. The 58-kDa polypeptide could also be eluted by chromanol and the 81-kDa polypeptide could be eluted also by phytol. The 81-kDa polypeptide had the unique P-E-E-D-Q-X-Q-Y N-terminal sequence.     

Erk1/2 MAPK and caldesmon differentially regulate podosome dynamics in A7r5 vascular smooth muscle cells

We tested the hypothesis that the MEK/Erk/caldesmon phosphorylation cascade regulates PKC-mediated podosome dynamics in A7r5 cells. We observed the phosphorylation of MEK, Erk and caldesmon, and their translocation to the podosomes upon phorbol dibutyrate (PDBu) stimulation, together with the nuclear translocation of phospho-MEK and phospho-Erk. After MEK inhibition by U0126, Erk translocated to the interconnected actin-rich columns but failed to translocate to the nucleus, suggesting that podosomes served as a site for Erk phosphorylation. The interconnected actin-rich columns in U0126-treated, PDBu-stimulated cells contained alpha-actinin, caldesmon, vinculin, and metalloproteinase-2. Caldesmon and vinculin became integrated with F-actin at the columns, in contrast to their typical location at the ring of podosomes. Live-imaging experiments suggested the growth of these columns from podosomes that were slow to disassemble. The observed modulation of podosome size and life time in A7r5 cells overexpressing wild-type and phosphorylation-deficient caldesmon-GFP mutants in comparison to untransfected cells suggests that caldesmon and caldesmon phosphorylation modulate podosome dynamics in A7r5 cells. These results suggest that Erk1/2 and caldesmon differentially modulate PKC-mediated formation and/or dynamics of podosomes in A7r5 vascular smooth muscle cells.     

Regulation of corticotropin-releasing factor receptor type 2beta mRNA via cyclic AMP pathway in A7r5 aortic smooth muscle cells

Corticotropin-releasing factor receptor type 2beta (CRF R2beta) is a member of the Class B heptahelical G protein-coupled receptors. This receptor is positively coupled to adenylate cyclase and is bound preferentially by the CRF-related peptides, urocortin (Ucn), Ucn II and Ucn III. In the rodent, CRF R2beta messenger RNA (mRNA) is expressed in the cardiovascular system, where its levels can be modulated by Ucn. In the present study, we investigated regulation of CRF R2beta levels by Ucn in A7r5 aortic smooth muscle cells. Ribonuclease protection assays show that A7r5 cells expressed the CRF R2beta subtype, which had two isoforms differing in one codon at the junction of exons 3 and 4. Ucn induced accumulation of intracellular cAMP via CRF R2beta in this cell line. In addition to the treatment with Ucn, cAMP agonists or analogues themselves caused a significant decrease in CRF R2beta mRNA levels. Blockade of Ucn- or cAMP-induced decreases in CRF R2beta mRNA levels by H7, a broad protein kinase inhibitor, suggested that a protein kinase pathway might be involved in this regulation. H89, a protein kinase A inhibitor, partially blocked Ucn- or cAMP-induced decreases in CRF R2beta mRNA levels. Thus, Ucn induces intracellular cAMP to downregulate CRF R2beta mRNA expression in A7r5 cells.     

PAK1 induces podosome formation in A7r5 vascular smooth muscle cells in a PAK-interacting exchange factor-dependent manner

Remodeling of the vascular smooth muscle cytoskeleton is essential for cell motility involved in the development of diseases such as arteriosclerosis and restenosis. The p21-activated kinase (PAK), which is an effector of the Rho GTPases Rac and Cdc42, has been shown to be involved in cytoskeletal remodeling and cell motility. We show herein that expression of cytoskeletally active constructs of PAK1 is able to induce the formation of dynamic, podosome-like F-actin columns in the A7r5 vascular smooth muscle cell line. Most of these actin columns appear at the junctions between stress fibers and focal adhesions and contain several known podosomal protein markers, such as cortactin, Arp2/3, -actinin, and vinculin. The kinase activity of PAK plays a role in the regulation of the turnover rates of these actin columns but is not essential for their formation. The ability of PAK to interact with the PAK-interacting exchange factor (PIX) but not with Rac or Cdc42, however, is required for the formation of the actin columns as well as for the translocation of PIX and G protein-coupled receptor kinase-interacting protein (GIT) to focal adhesions adjacent to the actin columns. These findings suggest that interaction between PAK and PIX, as well as the recruitment of PIX and GIT to focal adhesions, plays an important role in the formation of actin columns that resemble podosomes induced by phorbol ester in vascular smooth muscle cells. actin cytoskeleton; p21-activated kinase     

Ammonium chloride affects receptor number and lateral mobility of the vasopressin V2-type receptor in the plasma membrane of LLC-PK1 renal epithelial cells: role of the cytoskeleton

The acidotropic agent ammonium chloride (NH4Cl) not only affects receptor metabolism by inhibiting lysosomal acidification, but can also affect the targeting of proteins to specific membranes in polarized cells, possibly through effects mediated by the cytoskeleton. The present study examines the effects of NH4Cl and perturbers of cytoskeleton structure on vasopressin V2 receptor expression in LLC-PK1 renal epithelial cells. Surprisingly, long-term pretreatment of cells with NH4Cl or short-term treatment with the actin perturber cytochalasin B resulted in an up to 70% increase in specific Arg-8-vasopressin binding compared to control cells, which was independent of the presence of NH4Cl in the binding test, and apparently the result of increased V2 receptor expression. Perturbers of microtubules such as colchicine and vinblastine had no such effect. A rhodamine-labeled analog of vasopressin was used to fluorescently label the V2 receptor of LLC-PK1 cells, and microscopic measurements of membrane-localized fluorescence confirmed the increased V2 receptor expression in the basal plasma membrane subsequent to NH4Cl pretreatment. Lateral mobility of the V2 receptor was measured in living cells using the technique of microphotolysis (photobleaching). The fraction of mobile receptors was 0.2 in cells pretreated with NH4Cl, markedly reduced compared to that of 0.9 in untreated cells. The apparent lateral diffusion coefficient D was about 3 x 10(-10) cm2/s in both pretreated and untreated cells. Results for fluorescence labeling of the actin cytoskeleton indicate that NH4Cl pretreatment of LLC-PK1 cells results in perturbation of microfilament structure. All results imply that the cytoskeleton plays a central role in V2 receptor expression and lateral mobility.     

Regulation of capacitative and non-capacitative Ca2+ entry in A7r5 vascular smooth muscle cells

A capacitative Ca2+ entry (CCE) pathway, activated by depletion of intracellular Ca2+ stores, is thought to mediate much of the Ca2+ entry evoked by receptors that stimulate phospholipase C (PLC). However, in A7r5 vascular smooth muscle cells, vasopressin, which stimulates PLC, empties intracellular Ca2+ stores but simultaneously inhibits their ability to activate CCE. The diacylglycerol produced with the IP3 that empties the stores is metabolized to arachidonic and this leads to activation of nitric oxide (NO) synthase, production of NO and cyclic GMP, and consequent activation of protein kinase G. The latter inhibits CCE. In parallel, NO directly activates a non-capacitative Ca2+ entry (NCCE) pathway, which is entirely responsible for the Ca2+ entry that occurs in the presence of vasopressin. This reciprocal regulation of two Ca2+ entry pathways ensures that there is sequential activation of first NCCE in the presence of vasopressin, and then a transient activation of CCE when vasopressin is removed. We suggest that the two routes for Ca2+ entry may selectively direct Ca2+ to processes that mediate activation and then recovery of the cell.     

The regulation of the cytoplasmic free Ca2+ concentration in aortic smooth muscle cells (A7r5 line) after stimulation by vasopressin and bombesin

The characteristics of intracellular Ca2+ transient induced by vasopressin and bombesin in aortic smooth muscle cells were studied using flow cytometric analysis of indo-1 loaded cells. The two hormones induced a rapid and transient rise in [Ca2+]i. This Ca2+ transient was independent of the presence of extracellular Ca2+. Addition of bombesin to cells that have already been stimulated by vasopressin (or conversely the addition of vasopressin to bombesin-stimulated cells) results in a second Ca2+ transient that has a smaller amplitude. This transient is the same when the external Ca2+ concentration is lowered from 1.8 mM to 50 nM, suggesting that the agonist-sensitive pool reloaded using the Ca2+ that has been previously released into the cytoplasm. Intracellular Ca2+ pools that have been depleted by a prolonged incubation of the cells in a low Ca2+ medium can be refilled by shifting cells to a high Ca2+ medium. The reloading was analyzed in detail and found to be a slow process. It is hardly affected by Ni2+ or by (-)D888, a potent inhibitor of the voltage-dependent Ca2+ channels. It is accelerated when Ca2+ uptake by the Na+/Ca2+ exchange system is stimulated. The results suggest that Ca2+ homeostasis in aortic smooth muscle cells is achieved using mechanisms that are distinct from those operating in various acini and in striated muscles.     

Conventional protein kinase C mediates phorbol-dibutyrate-induced cytoskeletal remodeling in a7r5 smooth muscle cells

Phorbol dibutyrate (PDBu) induced the formation of podosome-like structures together with partial disassembly of actin stress fibers in A7r5 smooth muscle cells. These podosomes contained alpha-actinin, F-actin, and vinculin and exhibit a tubular, column-like structure arising perpendicularly from the bottom of PDBu-treated cells. The conventional protein kinase C (PKC) antagonist, GO6976, inhibited PDBu-induced cytoskeletal remodeling at 0.1 microM, whereas the novel PKC antagonist, rottlerin, was ineffective at 10 microM. PDBu induced the translocation of the conventional PKC-alpha but not the novel PKC-delta to the sites of podosome formation in A7r5 cells. Although partial disassembly of actin stress fibers was observed in both Y-27632- and PDBu-treated cells, focal adhesions were much reduced in number and size only in Y-27632-treated cells. Furthermore, PDBu restored focal adhesions in Y-27632-treated cells. Live video fluorescence microscopy of alpha-actinin GFP revealed a lag phase of about 20 min prior to the rapid formation and dynamic reorganization of podosomes during PDBu treatment. These findings suggest that conventional PKCs mediate PDBu-induced formation of dynamic podosome-like structures in A7r5 cells, and Rho-kinase is unlikely to be the underlying mechanism. The podosome columns could represent molecular scaffolds where PKC-alpha phosphorylates regulatory proteins necessary for Ca(2+) sensitization in smooth muscle cells.     

Stimulus-specific activation and actin dependency of distinct, spatially separated ERK1/2 fractions in A7r5 smooth muscle cells

A proliferative response of smooth muscle cells to activation of extracellular signal regulated kinases 1 and 2 (ERK1/2) has been linked to cardiovascular disease. In fully differentiated smooth muscle, however, ERK1/2 activation can also regulate contraction. Here, we use A7r5 smooth muscle cells, stimulated with 12-deoxyphorbol 13-isobutylate 20-acetate (DPBA) to induce cytoskeletal remodeling or fetal calf serum (FCS) to induce proliferation, to identify factors that determine the outcomes of ERK1/2 activation in smooth muscle. Knock down experiments, immunoprecipitation and proximity ligation assays show that the ERK1/2 scaffold caveolin-1 mediates ERK1/2 activation in response to DPBA, but not FCS, and that ERK1/2 is released from caveolin-1 upon DPBA, but not FCS, stimulation. Conversely, ERK1/2 associated with the actin cytoskeleton is significantly reduced after FCS, but not DPBA stimulation, as determined by Triton X fractionation. Furthermore, cytochalasin treatment inhibits DPBA, but not FCS-induced ERK1/2 phosphorylation, indicating that the actin cytoskeleton is not only a target but also is required for ERK1/2 activation. Our results show that (1) at least two ERK1/2 fractions are regulated separately by specific stimuli, and that (2) the association of ERK1/2 with the actin cytoskeleton regulates the outcome of ERK1/2 signaling.     

Oxysterols modulate calcium signalling in the A7r5 aortic smooth muscle cell-line

Prolonged exposure to oxidized low density lipoprotein (oxLDL) can alter various aspects of cell biology, including modification of vasomotor responses and downregulation of calcium channel proteins in aortic smooth muscle cells. However, the components of oxLDL responsible for these effects have not been fully elucidated. The study reported here aimed at examining the consequences of extended exposure to oxysterols, cholesterol oxidation products whose levels are elevated in oxLDL as compared to unmodified LDL, on calcium signalling mechanisms in A7r5 cells, a model aortic smooth muscle cell-line. Within 24 h of exposure, all three oxysterol congeners tested caused an elevation in the resting cytoplasmic Ca²⁺ concentration. These oxysterols also inhibited Ca²⁺ transients in response to arginine vasopressin and bradykinin, and some but not all congeners ablated Ca²⁺ signals triggered by platelet activating factor, the ryanodine receptor calcium channel agonist 4-choloro-meta-cresol, or thapsigargin, an inhibitor of endoplasmic reticulum Ca²⁺ uptake. The effects of long-term exposure to the oxysterol congener 7β-hydroxycholesterol on arginine vasopressin stimulated Ca²⁺ signals were mainly at the level of Ca²⁺ release from intracellular stores rather than on Ca²⁺ influx mechanisms. Of the calcium signalling proteins tested, only the type 1 ryanodine receptor and the type 1 inositol 1,4,5-trisphosphate receptor (IP₃R1) were significantly downregulated by 24 h exposure to oxysterols. Decreases in IP₃R1 protein triggered by 7β-hydroxycholesterol were both time and concentration dependent, occurring over a concentration range encountered within atherosclerotic lesions. IP₃R1 downregulation by certain oxysterols is mediated by proteasomal proteolysis, since it can be abolished by co-incubation with epoxomicin. Overall, these data demonstrate that major oxysterol components of oxLDL cause long-term alterations in Ca²⁺ signalling in a model aortic smooth muscle cell. Such effects could contribute to the pathology of atherosclerotic disease.     

TRPC6 is a candidate channel involved in receptor-stimulated cation currents in A7r5 smooth muscle cells

To investigate thepossible role of members of the mammalian transient receptor potential(TRP) channel family (TRPC1-7) in vasoconstrictor-inducedCa²⁺ entry in vascular smooth muscle cells, we studied[Arg⁸]-vasopressin (AVP)-activated channels in A7r5aortic smooth muscle cells. AVP induced an increase in free cytosolicCa²⁺ concentration ([Ca²⁺]_i)consisting of Ca²⁺ release and Ca²⁺ influx.Whole cell recordings revealed the activation of a nonselective cationcurrent with a doubly rectifying current-voltage relation strikinglysimilar to those described for some heterologously expressed TRPCisoforms. The current was also stimulated by direct activation of Gproteins as well as by activation of the phospholipase C-coupledplatelet-derived growth factor receptor. Currents were not activated bystore depletion or increased [Ca²⁺]_i.Application of 1-oleoyl-2-acetyl-sn-glycerol stimulated the current independently of protein kinase C, a characteristic property ofthe TRPC3/6/7 subfamily. Like TRPC6-mediated currents, cation currentsin A7r5 cells were increased by flufenamate. Northern hybridizationrevealed mRNA coding for TRPC1 and TRPC6. We therefore suggest thatTRPC6 is a molecular component of receptor-stimulated Ca²⁺-permeable cation channels in A7r5 smooth muscle cells.

     

cAMP-dependent protein kinase activation affects vasopressin V2-receptor number and internalization in LLC-PK1 renal epithelial cells.

The relationship between activation of the cAMP-dependent protein kinase (cAMP-PK) and ligand binding and internalization by the vasopressin renal (V2-type) receptor of LLC-PK1 renal epithelial cells was examined. Upon cAMP-PK activation through 1 h treatment with the cAMP analogue 8-bromo-cAMP (BrcA), a marked reduction in V2-receptor steady state number and internalization in LLC-PK1 cells was effected. In cells treated for 17 h with BrcA and hence down-regulated for cAMP-PK, the V2-receptor number was normal but internalization was markedly reduced. Cells of the LLC-PK1 mutant FIB4, which possesses about 10% parental cAMP-PK catalytic subunit activity, exhibited lower V2-receptor steady state number and internalization in comparison to untreated LLC-PK1 cells. A negative correlation was thus evident between cAMP-PK activation and V2-receptor number, and internalization. Phosphorylation by cAMP-PK may effect ligand-independent removal of receptor from the plasma membrane.