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Stress exposure is linked to elevated blood pressure, which increases risk for cardiovascular disease (Spruill, 2010; WHO, 2013). Stress exposure may be especially harmful when concentrated in one particular domain (i.e., low stressor diversity) (Koffer, et al., 2016). Using a diversity index, we test whether high stressor exposure and low stressor diversity is associated with high resting blood pressure. Participants (N=391, aged 40-64 years) completed four days of hourly self-report of stressful experiences (e.g., work task demand, non-work task demand, arguments, interpersonal tension), with clinic blood pressure separately assessed. Linear regression results indicate older adults experienced lower stressor diversity (B = -0.003, p =.003). Further, higher stressor exposure with lower stressor diversity related to higher diastolic blood pressure (B= -7.21, p=.046). Experiencing high stress concentrated in one domain may increase risk of high blood pressure. We discuss how low stressor diversity may help explain age-related risk of cardiovascular disease.

Domain-specific control beliefs buffer the influence stressors have on people’s negative affect (stressor-reactivity). However, little is known about the extent to which individuals’ daily control beliefs are spread across domains (i.e., control diversity) and whether control diversity is adaptive. To examine these questions, we apply multilevel modeling to daily diary data from the National Survey of Daily Experiences (N=2,022; mean age=56; 33–84; 57% women). Results indicate that above and beyond average control, people with more control diversity reported more negative affect and evidenced greater stressor reactivity compared to people with less control diversity. Although older adults’ control is spread across fewer domains than middle-aged and younger adults’ control, there was no evidence of age-differences in how control diversity relates to negative affect and stressor reactivity. Findings suggest that daily control diversity provides unique insights into how control beliefs change across adulthood and old age and contribute to affective well-being.

The transmembrane adaptor protein NTAL (non-T-cell activation linker) participates in signalosome assembly in hematopoietic cells, but its exact role in cell physiology remains enigmatic. We report here that BM-derived mast cells from NTAL-deficient mice, responding to Ag alone or in combination with SCF, exhibit reduced spreading on fibronectin, enhanced filamentous actin depolymerization and enhanced migration towards Ag relative to WT cells. No such differences between WT and NTAL(-/-) BM-derived mast cells were observed when SCF alone was used as activator. We have examined the activities of two small GTPases, Rac and Rho, which are important regulators of actin polymerization. Stimulation with Ag and/or SCF enhanced activity of Rac(1,2,3) in both NTAL(-/-) and WT cells. In contrast, RhoA activity decreased and this trend was much faster and more extensive in NTAL(-/-) cells, indicating a positive regulatory role of NTAL in the recovery of RhoA activity. After restoring NTAL into NTAL(-/-) cells, both spreading and actin responses were rescued. This is the first report of a crucial role of NTAL in signaling, via RhoA, to mast cell cytoskeleton.

Increasing cellular G-actin, using latrunculin B, in either intact or permeabilized rat peritoneal mast cells, caused translocation of both actin and an actin regulatory protein, cofilin, into the nuclei. The effect was not associated with an increase in the proportion of apoptotic cells. The major part of the nuclear actin was not stained by rhodamine-phalloidin but could be visualized with an actin antibody, indicating its monomeric or a conformationally distinct state, e.g. cofilin-decorated filaments. Introduction of anti-cofilin into permeabilized cells inhibited nuclear actin accumulation, implying that an active, cofilin-dependent, import exists in this system. Nuclear actin was localized outside the ethidium bromide-stained region, in the extrachromosomal nuclear domain. In permeabilized cells, the appearance of nuclear actin and cofilin was not significantly affected by increasing [Ca(2+)] and/or adding guanosine 5'-O-(3-thiotriphosphate), but was greatly promoted when ATP was withdrawn. Similarly, ATP depletion in intact cells also induced nuclear actin accumulation. In contrast to the effects of latrunculin B, ATP depletion was associated with an increase in cortical F-actin. Our results suggest that the presence of actin in the nucleus may be required for certain stress-induced responses and that cofilin is essential for the nuclear import of actin.

Many proline-rich proteins participate in delivering actin monomers to specific cellular locations where actin-rich membrane protrusions, such as ruffles, filopodia and microspikes, are formed. These protrusions are necessary for cell motility. Actin monomer is usually delivered to the site of polymerization in the form of profilactin - a complex of G-actin with a polyproline-binding protein, profilin. Here, we describe proline-rich proteins involved in regulating actin polymerization and classify them according to their role in recruiting profilin to the membrane.

Secretion is dependent on a rise in cytosolic Ca(2+)concentration and is associated with dramatic changes in actin organization. The actin cortex may act as a barrier between secretory vesicles and plasma membrane. Thus, disassembly of this cortex should precede late steps of exocytosis. Here we investigate regulation of both the actin cytoskeleton and secretion by calmodulin. Ca(2+), together with ATP, induces cortical F-actin disassembly in permeabilized rat peritoneal mast cells. This effect is strongly inhibited by removing endogenous calmodulin (using calmodulin inhibitory peptides), and increased by exogenous calmodulin. Neither treatment, however, affects secretion. Low concentrations ( approximately 1 microM) of a specific inhibitor of myosin light chain kinase, ML-7, prevent F-actin disassembly, but not secretion. In contrast, a myosin inhibitor affecting both conventional and unconventional myosins, BDM, decreases cortical disassembly as well as secretion. Observations of fluorescein-calmodulin, introduced into permeabilized cells, confirmed a strong (Ca(2+)-independent) association of calmodulin with the actin cortex. In addition, fluorescein-calmodulin enters the nuclei in a Ca(2+)-dependent manner. In conclusion, calmodulin promotes myosin II-based contraction of the membrane cytoskeleton, which is a prerequisite for its disassembly. The late steps of exocytosis, however, require neither calmodulin nor cortical F-actin disassembly, but may be modulated by unconventional myosin(s).

To investigate the role of the actin cytoskeleton in exocytosis, we have tested the effects of latrunculin B, a microfilament-disrupting drug, on secretion from intact and permeabilised rat peritoneal mast cells. The toxin strongly inhibited secretion from intact cells (attached or in suspension) responding to a polybasic agonist, compound 48/80. However, this effect was revealed only after a profound depletion of actin filaments. This was achieved by a long (1 h) exposure of cells to the drug before activation, together with its presence during activation. Maximal inhibition of secretion by such treatment was 85% at 40 microgram/ml latrunculin B. These results indicate that minimal actin structures are essential for the exocytotic response. In contrast, stimulus-induced cell spreading was prevented by latrunculin (5 microgram/ml) applied either before or after activation. The effects of the toxin on intact cells were fully reversible. The responses of permeabilised cells were affected differentially: secretion induced by calcium was more sensitive to latrunculin than that induced by GTP-gamma-S. The calcium response, therefore, is more dependent upon the integrity of the actin cytoskeleton than the response induced by GTP-gamma-S. Again, maximal inhibitory effects (approximately 65 and 25% at 40 microgram/ml) were observed only when cells were exposed to the toxin both before and after permeabilisation. Since the permeabilised cells system focuses on the final steps of exocytosis, the incomplete inhibition suggests that actin plays a modulatory rather than a central role at this stage.

Using a probe obtained by PCR amplification from mouse genomic DNA, a genomic clone was isolated covering the entire mouse preprogalanin gene. The mouse gene has an exon:intron organisation very similar to that of the rat and human genes. The first exon is noncoding while exons 2-5 carry the coding region. Exon 6 also encodes the stop codon and a polyadenylation signal. The deduced amino-acid sequence of mouse preprogalanin is 94% and 68% identical to the rat and human peptide, respectively. The amino-acid sequence of mouse galanin was confirmed by RT-PCR amplification of mouse brain RNA. The cloning of the mouse galanin gene should allow elucidation of the regulatory characteristics of its promoter and facilitate transgenic approaches to the analysis of galanin gene function in this species.

Rat peritoneal mast cells, both intact and permeabilized, have been used widely as model secretory cells. GTP-binding proteins and calcium play a major role in controlling their secretory response. Here we have examined changes in the organization of actin filaments in intact mast cells after activation by compound 48/80, and in permeabilized cells after direct activation of GTP-binding proteins by GTP-gamma-S. In both cases, a centripetal redistribution of cellular F-actin was observed: the content of F-actin was reduced in the cortical region and increased in the cell interior. The overall F-actin content was increased. Using permeabilized cells, we show that AIF4-, an activator of heterotrimeric G proteins, induces the disassembly of F-actin at the cortex, while the appearance of actin filaments in the interior of the cell is dependent on two small GTPases, rho and rac. Rho was found to be responsible for de novo actin polymerization, presumably from a membrane-bound monomeric pool, while rac was required for an entrapment of the released cortical filaments. Thus, a heterotrimeric G-protein and the small GTPases, rho and rac, participate in affecting the changes in the actin cytoskeleton observed after activation of mast cells.

Cells used in this work were permeabilized by streptolysin-O and then washed to remove freely soluble components. The secretory responsiveness of these cells to various combinations of calcium, MgATP and guanine nucleotide was characterized and in most respects was found to be similar to that of the metabolically inhibited (unwashed) cells. The content of adenosine and guanine nucleotides, which remain within the permeabilized cells after washing, was estimated as 0.83 and 0.12 mM (extrapolated to intact cells), which constitutes 18 and 25%, respectively, of the total nucleotide content of mast cells. High (> mM) concentrations of MgATP, required for the calcium-induced secretion, were reduced to μM levels by suboptimal concentrations of GTP, which also markedly increased both the rate and extent of the response. Similarily, μM concentrations of MgATP reduced the requirements of the calcium-dependent secretion for GTP. The synergy of the GTP and ATP effects suggests that, together, the two nucleotides can maintain a pool of free GTP, presumably as a result of transphosphorylation from ATP to GDP. Thus, MgATP may work by transphosphorylating the endogenous GDP. However, neither GTP nor GTP-γ-S were effective as substitutes for MgATP in the calcium-induced secretion, particularly that from metabolically inhibited cells. This indicates that MgATP does not act simply by providing GTP but is needed to maintain a phosphorylated state of the system. The synergistic effects of ATP and GTP were observed only in the presence of calcium. To test whether calcium/MgATP-induced secretion requires an activated G protein, the effects of G-protein inactivators were studied. GDP, deoxy GDP and GDP-β-S exerted differing degrees of inhibition on secretory responses induced by various combinations of effectors. The response to calcium/MgATP was less sensitive to these inhibitors than that to GTP-γ-S (with or without calcium). However, all three ‘inhibitors’ were also capable of stimulating calcium/MgATP-dependent secretion, indicating a transphosphorylation, producing GTP, dGTP and GTP-β-S. Thus, in the absence of any specific inhibitors for either G proteins or the transphosphorylation reaction, the degree of dependence of the calcium-induced secretion on a G protein remains unclear.

Treatment with metabolic inhibitors and addition of exogenous MgATP exerted different effects on secretion from streptolysin-O-permeabilized mast cells, responding to calcium and GTP-gamma-S as single effectors (i.e., independently of each other). These effects were also strongly dependent on the experimental conditions. Thus cells, triggered by Ca2+ at the time of permeabilization, did not require MgATP, but after metabolic inhibition rapidly became absolutely dependent on its provision, requiring high (mM) concentrations. AMP-PNP was not effective. After longer treatment with metabolic inhibitors, the absolute dependence on MgATP was also exhibited by cells responding to dual effectors (i.e., Ca2+ and GTP-gamma-S applied together). In contrast, calcium independent secretion due to GTP-gamma-S was more resistant to metabolic inhibition, exhibiting no absolute requirements for MgATP. Once the responsiveness to GTP-gamma-S had been lost, it could not be restored by addition of MgATP. MgATP, in fact, inhibited the response of permeabilized cells to GTP-gamma-S. This effect could be mimicked by AMP-PNP. When permeabilized cells were washed before triggering, MgATP (0.1-1 mM concentration range) was no longer inhibitory but stimulatory. These differences between Ca(2+)- and GTP-gamma-S-induced responses indicate that ATP utilization is essential to the calcium, but not to the guanine nucleotide, pathway to secretion. The rate of the response to calcium/MgATP was much slower in the absence than in the presence of GTP-gamma-S. The onset of secretion occurred after an initial delay. This lag phase was abolished by addition of GTP-gamma-S, suggesting that a GTP-binding protein may control a reaction which constitutes a rate-limiting step in the secretory process.

The major part of mast cell actin is Triton-soluble and behaves as a monomer in the DNase I inhibition assay. Thus, actin exists predominantly in monomeric or short filament form, through filamentous actin is clearly apparent in the cortical region after rhodamine-phalloidin (RP) staining. The minimum actin content is estimated to be approximately 2.5 micrograms/10(6) cells (cytosolic concentration approximately 110 microM. After permeabilization of mast cells by the bacterial cytolysin streptolysin-O, approximately 60% of the Triton-soluble actin leaks out within 10 min. However, the staining of the cortical region by RP remains undiminished, and the cells are still capable of exocytosis when stimulated by GTP-gamma-S together with Ca2+. In the presence of cytochalasin E the requirement for Ca2+ is decreased, indicating that disassembly of the cytoskeleton may be a prerequisite for exocytosis. This disassembly is likely to be controlled by Ca2(+)-dependent actin regulatory proteins; their presence is indicated by a Ca2(+)-dependent inhibition of polymerization of extraneous pyrene-G-actin by a Triton extract of mast cells. The effect of cytochalasin E on secretion is similar to that of phorbol myristate acetate, an activator of protein kinase C; both agents enhance the apparent affinity for Ca2+ and cause variable extents of Ca2(+)-independent secretion. Exposing the permeabilized cells to increasing concentrations of Ca2+ caused a progressive decrease in F-actin levels as measured by flow cytometry of RP-stained cells. In this respect, both cytochalasin E and phorbol ester mimicked the effects of calcium. GTP-gamma-S was not required for the Ca2(+)-dependent cortical disassembly. Thus, since conditions have not yet been identified where secretion can occur in its absence, cortical disassembly may be essential (though it is not sufficient) for exocytosis to occur.

Mast cells reorganize their actin cytoskeleton in response to cytosolic calcium signals while in parallel secreting histamine and other inflammatory mediators. The effect of calcium on actin is mediated in large part through calmodulin. EGFP-tagged calmodulin is concentrated in the actin-rich cortex of RBL-2H3 mast cells. Transfection with small interfering RNA directed against the actin and calmodulin-binding protein IQGAP1 dramatically reduced expression of the latter protein and reduced or eliminated the concentration of calmodulin at the actin-rich cortex. Both actin reorganization and secretion were enhanced in IQGAP1 knockdown cells. Our results suggest a model in which calmodulin is targeted to and sequestered at the actin cytoskeleton by IQGAP1. Upon cell stimulation and the subsequent [Ca2+]i increase, it is immediately available to activate local downstream targets.

A line of rat basophilic leukaemia (RBL) cells, a model of mast cells, stably expressing EGFP-tagged calmodulin secreted normally in response to standard agonists. As reported for other cell types, calmodulin was concentrated in the mitotic spindle poles of dividing cells. In unstimulated interphase cells calmodulin was concentrated in the cell cortex and at a single central location. Disruption of cortical actin eliminated the concentration of calmodulin at the cortex while the central calmodulin concentration was associated with an enrichment of tubulin and is likely to represent the centrosome. Following stimulation with either an agonist that crosslinks Fc receptors or co-application of phorbol ester and a calcium ionophore the interior of the cells lost calmodulin while cortical fluorescence became more pronounced but also less uniform. After stimulation discrete bright puncta of calmodulin-EGFP (CaM-EGFP) appeared in the cell interior. Puncta colocalised with moving lysotracker-labelled granules, suggesting that calmodulin may play a role in organising their transport. Our results show that in interphase RBL cells a large fraction of the calmodulin pool is associated with targets in the actin cytoskeleton and demonstrate the utility of this model system for studying calmodulin biology.

Publisher Summary This chapter describes the Rho guanosine 5'-triphosphates (GTPases) secretion and actin dynamics in permeabilized mast cells. Permeabilized mast cells proved to be a valuable tool for studying the function of Rho-related GTPases. Rho, Rac, and Cdc42 were shown to be crucial for regulation of the late steps of exocytosis. The chapter discusses the use of primary mast cells—permeabilized by streptolysin O (SLO)—for studying Rho family GTPase. The chapter discusses the preparation of recombinant GTPases, an assay for GTP-binding activity, and subsequent loading of recombinant proteins with GTP. The chapter describes various procedures for introducing GTPase into mast cells and evaluation of their effects on the cytoskeleton and exocytosis. The chapter also discusses the isolation, permeabilization, and stimulation of rat peritoneal mast cells. The chapter concludes with a discussion of the analysis of F-actin content and morphology.

In mast cells, activation of GTP-binding proteins induces centripetal reorganization of actin filaments. This effect is due to disassembly, relocalization, and polymerization of F-actin and is dependent on two small GTPases, Rac and Rho. Activities of Rac and Rho are also essential for the secretory function of mast cells. In response to GTP-gamma-S and/or calcium, only a proportion of permeabilized mast cells is capable of secretory response. Here, we have compared actin organization of secreting and nonsecreting cell populations. We show that the cytoskeletal and secretory responses are strongly correlated, indicating a common upstream regulator of the two functions. The secreting cell population preferentially displays both relocalization and polymerization of actin. However, when actin relocalization or polymerization is inhibited by phalloidin or cytochalasin, respectively, secretion is unaffected. Moreover, the ability of the constitutively active mutants of Rac and Rho to enhance secretion is also unaffected in the presence of cytochalasin. Therefore, Rac and Rho control these two functions by divergent, parallel signaling pathways. Cortical actin disassembly occurs in both secreting and nonsecreting populations and does not, by itself, induce exocytosis. A model for the control of exocytosis is proposed that includes at least four GTP-binding proteins and suggests the presence of both shared and divergent signaling pathways from Rac and Rho.

Permeabilised rat mast cells were exposed to gelsolin and its N-terminal half (S1-3), proteins that sever actin filaments in a calcium-dependent and independent manner, respectively. Gelsolin and S1-3 induced a decrease in cellular F-actin content and an increase in the extent of the secretory response. The calcium sensitivities of both these effects were consistent with the differential calcium requirements of the two proteins. Segment 1 (S1), which binds G-actin and caps filaments but does not sever them, did not show these effects. Thus, secretion of mast cells is promoted as a consequence of the severing activity of exogenous gelsolin or S1-3. Most of the endogenous gelsolin remained within permeabilised, washed mast cells and its distribution in resting state was predominantly cortical. Addition of calcium in the absence of MgATP did not reduce the F-actin content; by contrast, calcium with MgATP induced F-actin loss that was unaffected by the presence of anti-gelsolin. Because this antibody inhibits the severing activity of gelsolin, these results indicate that in permeabilised mast cells the severing activity of the remaining endogenous gelsolin is not involved in cortical actin filaments disassembly. Upon exposure to GTP-gamma-S in the absence of calcium, the content of cortical gelsolin was reduced. This parallels our previous observation of a GTP-gamma-S induced reduction of cortical actin filaments followed by their relocation to the cell's interior (Norman et al. (1994) J. Cell Biol. 126, 1005–1015) and suggests that actin redistribution may be a consequence of dissociation of gelsolin caps brought about by activation of a GTP-binding protein.

The release of inflammatory mediators from mast cells occurs by a regulated exocytotic process. We have been able to study the intracellular events in this pathway by permeabilising the plasma membrane of rat peritoneal mast cells and stimulating exocytosis by providing both Ca2+ and a guanine nucleotide. By this approach we have obtained evidence for the participation of at least two guanine nucleotide binding proteins in the control of exocytosis. We have also shown that ATP is unnecessary for the final events, but that it does have a number of modulatory functions, for instance in the control of the effective affinity of the proteins that bind Ca2+ and GTP. There is also evidence for a protein dephosphorylation in the later stages of the control pathway.

Plasma membranes, isolated from normal (C13) and polyoma virus-transformed (J1) cultured BHK cells were incubated with G-actin under polymerizing conditions, followed by a low-speed centrifugation. The amount of actin attached to the pelleted BHK-J1 plasma membranes was at least twice that on BHK-C13 membranes, indicating a greater number of actin attachment sites on the former. This result was confirmed by the observation that the plasma membranes from the transformed cells were also more active in nucleating polymerization of pyrene-labelled actin. Most of the actin attachment sites could be solubilized by Triton or low-salt extraction treatment.

Activation of skeletal muscle myosin and myosin subfragment-1 (S1) by actin purified from the cytoplasm of cultured BHK cells was studied using the fluorescence of pyrene-labelled BHK F-actin and its quenching by S1 and by an enzyme-linked ATPase assay. At non-saturating concentrations, both muscle and BHK actin activated skeletal muscle myosin to the same degree: at 30 degrees C and an ionic strength of 108 mM, 1 microM actin approximately doubled the ATPase of myosin or of S1. The association between BHK actin and S1 was also followed in a fluorescence stop flow: the rate of ATP binding monitored by the loss of light scattering upon dissociation of actin was again the same for BHK and muscle actin. The similarity of activation of myosin ATPase by BHK and muscle actin at low actin concentrations (i.e. the similarity of Vmax/Km) suggests that both Vmax and Km are similar for the two types of actin. The effect of varying filament length on actin activation of myosin ATPase was examined using pig plasma or BHK gelsolin to regulate the length. For both types of actin, maximum enhancement of the actomyosin ATPase activity was observed at an actin/gelsolin ratio of about 30:1, whereas inhibition was observed at lower ratios. Both activation and inhibition of actomyosin ATPase were apparent in the absence or presence of calcium; differences were observed only in the extent and the time course of the effect.

Sudden symptomatic increases in blood pressure may result from withdrawal of antihypertensive drug therapy. Captopril, an orally active angiotensin converting enzyme inhibitor, was abruptly withdrawn from the antihypertensive regimen of 6 patients (5 males; aged 31 to 58) with moderate to severe hypertension. The patients had received captopril (50-450 mg/day), diuretics, and in 2 cases, propranolol for 3-13 months; all drugs other than captopril were continued during the study. Blood pressure increased gradually in all patients from a mean (+/- SEM) of 135/92 +/- 5/2 mm Hg to 164/111 +/- 8/3 mm Hg in the 14 to 156 hours following captopril withdrawal, without the occurrence of symptoms. No "rebound" increase in blood pressure was noted. Plasma renin activity decreased, plasma aldosterone and serum angiotensin II concentrations increased and plasma catecholamine concentrations did not change during this time. These changes are consistent with the cessation of angiotensin converting enzyme inhibition. Reinstitution of captopril reduced blood pressure to the previous nadir suggesting a lack of tolerance to its effects after chronic therapy.

Cytoplasmic actin from cultured fibroblasts has been purified to homogeneity and characterized with respect to its polymerization and structure. It was found to be qualitatively similar to muscle actin in all respects, but significant quantitative differences in its properties were demonstrated. Although BHK actin did not polymerize in unfractionated cytoplasmic extracts, the purified BHK actin polymerized into filaments both in magnesium and calcium. The critical concentration, measured by the DNase I inhibition assay and by fluorimetry, was the same as that of muscle actin both in magnesium and calcium. Polymerization of pyrene-labelled BHK and muscle actin was followed by fluorimetry. Significant differences in kinetics were found under both ionic conditions tested. In the absence of Mg2+ ions (0.2 mM CaCl2, 85 mM KCl), BHK actin polymerized at a much slower rate than muscle actin. In the presence of magnesium and EGTA, the nucleation phase for BHK actin polymerization was shorter than that for muscle actin and the kinetics of polymerization was different. The structure of BHK actin filaments in the electron micrographs was very similar to that of muscle actin. In high concentrations of magnesium, BHK actin formed paracrystals which had the same appearance as muscle actin paracrystals. However, calcium-induced formation of actin paracrystals required higher concentration of Ca2+ ions for BHK actin than for muscle actin (12 mM and 8 mM respectively). These results suggest differences in divalent cation binding to both high- and low-affinity sites of the two actins.

High-speed supernatant obtained from the lysate of cultured BHK cells has been chromatographed on Sepharose-4B, DEAE-cellulose and hydroxyapatite columns, and a fraction has been identified with characteristics similar to an actin depolymerizing factor (ADF), a small protein previously isolated from embryonic chick brain. Using a rabbit antibody against the chick brain protein, two immunoreactive forms were identified: a 19 kDa form co-migrating in SDS-polyacrylamide gels with embryonic chick brain ADF, and a 20 kDa form. The two species could be separated on a hydroxyapatite or green A dye matrix columns and only the 20 kDa protein was active when assayed for effects on pyrene-G-actin assembly. It enhanced the rate of F-actin assembly, but only after an initial lag phase, and decreased the final proportion of actin in filamentous form. These effects were calcium-independent. Actin depolymerizing factor constituted at least 0.5% of the total protein in the cytoplasmic fraction. A Triton extract of plasma membrane-enriched fraction from BHK cells was fractionated on a Sepharose-4B column and again, a fraction was found which had an ADF-like activity and also contained the two immuno-cross-reactive forms, 19 kDa and 20 kDa. These results suggest a novel regulation of the microfilament system in eukaryotic cells via the control of the ADF activity.

This study addresses the question of the role of cytoplasmic proteins in exocytosis from permeabilised rat mast cells. We have used two different methods of cell permeabilisation (ATP4- and streptolysin O) to regulate the size of the plasma membrane lesions, and thus to dictate the rate and extent of efflux of the cytosolic proteins, and compared the secretory response of the two preparations. We report evidence for the existence of two factors present in the cytosol, which affect the exocytotic mechanism in opposing manners. One of these is required for the maintenance of cell responsiveness; it is retained for more than 120 min by ATP4- -permeabilised cells but lost within 60 min from cells permeabilised by streptolysin O. The other factor, which leaks immediately from cells treated from streptolysin O, but only gradually from cells treated with ATP4-, has the effect of suppressing the affinity for both Ca2+ and guanine nucleotide in the exocytotic reaction.

The actin in cold lysates of cultured baby hamster kidney cells was predominantly monomeric, as judged by the DNase inhibition assay. Most of the filamentous actin attached to the membranes could be dissociated by washing with low ionic strength buffer, but a minor part was tightly associated in complexes with other proteins and could not be liberated for assay by DNase after exposure to a depolymerizing medium. The monomeric actin in the cell extract must be sequestered as a complex with a profilin-like protein, since its concentration was greatly in excess of the critical concentration for polymerization. On warming, rapid polymerization ensued, with loss of most of the monomeric actin. A separate cross-linking process could be observed by light-scattering measurements. A gel was formed, that contained besides actin substantial amounts of constituents identified as filamin, myosin, a-actinin, a gelsolin-like protein and other species of apparent subunit molecular weights 58 000 and 300 000, presumed to derive from intermediate filaments. Some low molecular weight protein subunits, including tropomyosin, were also present. The gelation was not accompanied by a significant degree of phosphorylation of any of the actin-associated proteins, with the exception of the minor species of 300000 molecular weight, the phosphorylation of which was cAMP-dependent. In the presence of concentrations of free calcium in the sub-micromolar range, the gel initially formed contracted and gradually dispersed. At higher concentrations of free calcium a loose turbid gel formed, but the bulk of the actin was found in the supernatant after high-speed centrifugation. Using a fluorescent actin derivative as a tracer, the quantum yield of which increases by nearly two orders of magnitude when it is incorporated into a polymer, it was established that the actin in the supernatant was not monomeric, but rather in the form of very short filaments. It was shown that scission of filaments accompanied the contraction of the cytoplasmic gel at the lower concentrations of calcium. The short filaments were highly effective in nucleating the polymerization of added G-actin and were evidently rapidly dissociated by DNase, since they reacted as monomers in the DNase inhibition assay. The gel obtained in the absence of calcium was revealed by electron microscopy to consist of clusters of filaments, many of which show a highly regular lateral spacing of about 7·0 nm. The physical basis for the temperature- and calcium-dependent phase equilibrium observed in this system are analysed in semi-quantitative terms.

This study evaluated the effects of single doses of the angiotensin converting enzyme inhibitor captopril and the beta-adrenergic blocking agent propranolol, alone or in combination, on the blood pressure, heart rate and humoral responses to both isometric (handgrip) and dynamic (ergometric) exercise in normotensive and hypertensive men. Single oral doses of either placebo, captopril 50 mg, propranolol 80 mg, or the latter two in combination were administered to age-matched groups (n = 5) of normotensive and hypertensive men in a random, double-blind manner. Captopril alone was indistinguishable from placebo after both isometric and ergometric exercise. Propranolol suppressed heart rate after both types of exercise and tended to decrease systolic blood pressure only in the hypertensive group; combination with captopril did not alter these responses. These data suggest that in sodium-replete subjects undergoing short-term vigorous exercise, the renin-angiotensin system, as measured by captopril inhibition, is less important than the sympathoadrenal system, as measured by propranolol inhibition, in the reflex cardiovascular adjustments accompanying acute isometric and dynamic exercise in both normotensive and hypertensive men.

Background: Regulated secretion by mast cells is known to be controlled by GTP-binding proteins, but the proteins involved have not been identified. Rac and Rho, two small GTPases related to the oncoprotein Ras, mediate transmission of signals from cell-surface receptors to the actin cytoskeleton. In rat mast cells, both Rac and Rho participate in effecting the centripetal redistribution of filamentous actin that is observed after stimulation of the cells. Rho is responsible for polymerization of actin filaments in the cell interior, whereas Rac is required for the entrapment in the interior of filaments released from the cortex. Such cytoskeletal changes could be important in control of the exocytotic process, so we examined whether Rac and Rho also play a role in regulated secretion by mast cells. Results We show that the constitutively active mutant proteins, V14RhoA and V12Rac1, enhance regulated secretion from permeabilized mast cells by increasing the proportion of cells that are competent to respond to stimulation. In addition, inhibition of endogenous Rac and Rho activity using inhibitors, N17Rac1 and C3 transferase, respectively, reduces the secretory response of mast cells to stimuli. Conclusion These results provide direct evidence that, in mast cells, both Rac and Rho are components of the signalling pathway that leads to secretion.

1. Nuclei isolated from red and white skeletal muscle, heart, uterus and liver of rabbit were incubated with [lambda-32P]ATP and the incorporation of 32P into nuclear protein was examined. 2. Liver nuclear proteins were phosphorylated to a much greater extent than those of muscle. Particularly low levels of phosphorylation were observed in white greater extent than those of muscle. Particularly low levels of phosphorylation were observed in white muscle nuclei. 3. Most of the phosphoproteins were common to all the tissues but significant quantitative differences and a limited number of qualitative differences were evident, particularly between liver and the muscle tissues. 4. Many phosphoproteins detected were minor components of the total nuclear protein.

The content and state of actin in baby hamster kidney (BHK) cells before and after transformation with polyoma virus were examined by deoxyribonuclease assay and gel electrophoresis followed by dye elution. The actin content of the transformed cells, relative to total cell protein, was lower than that of the normal cells by 30–50%. In both the normal and transformed cells the greater part of the total actin was found on lysis to be in the monomeric state. Cytoplasmic and membrane fractions of the two cell lines were, in qualitative terms, very similar in their protein compositions. The plasma membrane isolated from the transformed cells was richer in actin than that from the untransformed, and both membrane fractions contained proteins corresponding to myosin, filamin and α-actinin on SDS-polyacrylamide gels. The cell extract from both the normal and transformed lines formed an actin-based gel on incubation at 30 °C, although the amount of the cross-linked actin was much smaller in the latter. This was a consequence not only of the lower concentration of total actin in the cell, but also, presumably, of a gross relative deficiency in the concentration or activity of filament cross-linking protein(s) in the cytoplasm. Thus, small aliquots of cytoplasmic fractions from transformed cells, when added to an excess of exogenous F-actin, were able to cross-link the filaments to a much smaller extent than those from the normal cells. A similar range of proteins was found to be associated with the actin gels formed from both cell extracts. One conspicuous difference was that a species migrating in SDS-gel electrophoresis as a doublet with a subunit molecular weight of about 58,000, and tentatively identified as intermediate filament protein, was replaced in the transformed cells by a single band. Filament cross-linking activity of the cytoplasmic fractions was enhanced by addition of Triton extracts of crude membranes, although the latter were not capable of cross-linking exogenous F-actin on their own. The effect of Triton extracts was much greater in the case of membranes from the transformed cells. The cytoplasmic fractions of BHK cells contain capping protein(s) and/or complexes of such proteins with actin; these reveal themselves by the propensity of the extracts to nucleate polymerization of exogenous G-actin. This activity was more abundant in transformed cells, despite their lower actin content. Their membranes were also more effective in nucleating G-actin polymerization, indicating the presence of a greater number of filament ends. These various differences between normal and transformed cells were in no case related to differences in state of growth, for they could not be discerned in comparisons of materials derived from quiescent and growing normal BHK cells.

A large proportion (60-85%) of the actin present in lysates of cultured baby hamster kidney (BHK) cells is monomeric at low temperature. All this G-actin is accounted for in the fractions obtained by high-speed centrifugation of the lysate in the presence or absence of non-ionic detergent; 98 (+/− 7)% of its reacts as monomer in the deoxyribonuclease assay. The fractions nevertheless possess actin polymerization nucleating activity, as assayed by the fluorescence of extraneous pyrene-conjugated actin, reflecting the presence of capping proteins and their complexes with oligomeric actin, which dissociate during the deoxyribonuclease assay. Despite the enhanced nucleation, the total proportion of actin polymerized at equilibrium is reduced, relative to that of actin in the absence of cell extract, presumably because of the presence of ‘barbed’-end capping protein(s). With increasing concentrations of the supernatant material, the proportion of polymerized actin was progressively reduced and the presence of inhibitory activity against polymerization became apparent. On addition of micromolar concentrations of free calcium, nucleation by the supernatant fractions is greatly accelerated, but the extent of polymerization undergoes a further reduction, due to the elevated critical monomer concentration resulting from capping. Inhibition of polymerization by the supernatant fraction in the presence of calcium is much greater than by saturating concentrations of cytochalasin E. These observations are most simply interpreted as showing that the cytoplasm additionally contains a ‘pointed’-end capping protein. The plasma membranes were found to have nucleating activity largely residing in the Triton-insoluble fractions. Actin cross-linking activity was assayed by adding small proportions of either the supernatant or membrane fractions to a large excess of F-actin and analysing the pellet resulting from a low-speed centrifugation. Supernatant fraction, containing Triton-soluble membrane-derived material, possessed much greater cross-linking capacity than the membrane-free supernatant prepared in the absence of detergent. The membrane fractions had practically no cross-linking activity on their own. Nevertheless, a non-ionic detergent extract of the membrane enhanced the cross-linking activity of the supernatant, indicating the participation of more than one species in a cross-linking process.