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Although it is known that mechanical stretching of cells can induce significant increases in cell growth and shape, the intracellular signaling pathways that induce this response at the level of the cell nucleus is unknown. The transport of molecules from the cell cytoplasm to the nucleoplasm through the nuclear pore is a key pathway through which gene expression can be controlled in some conditions. It is presently unknown if mechanical stimuli can induce changes in nuclear pore expression and/or function. The purpose of the present investigation was to determine if mechanical stretching of a cell will alter nuclear protein import and the mechanisms that may be responsible. Vascular smooth muscle cells that were mechanically stretched exhibited an increase in proliferating cell nuclear antigen expression, cell number, and cell size within 24-48 h. Cells were microinjected with marker proteins for nuclear import. Nuclear protein import was significantly stimulated in stretched cells when compared with control. This was associated with an increase in the expression of nuclear pore proteins as detected by Western blots. Inhibition of the MAPK pathway blocked the stretch-induced stimulation of both cell proliferation and nuclear protein import. We conclude that nuclear protein import and nuclear pore density can adapt to mechanical stimuli during the process of cell growth through a MAPK-mediated mechanism.

Lysophosphatidylcholine (lyso-PC) is a product of phosphatidylcholine hydrolysis by phospholipase A2 (PLA2) and is present in cell membranes, oxidized lipoproteins, and atherosclerotic tissues. It has the ability to alter endothelial functions and is regarded as a causal agent in atherogenesis. In this study, the modulation of arachidonate release by lyso-PC in human umbilical vein endothelial cells was examined. Incubation of endothelial cells with lyso-PC resulted in an enhanced release of arachidonate in a time- and concentration-dependent manner. Maximum arachidonate release was observed at 10 min of incubation with 50 microM lyso-PC. Lyso-PC species containing palmitoyl (C16:0) or stearoyl (C18:0) groups elicited the enhancement of arachidonate release, while other lysolipids such as lysophosphatidylethanolamine, lysophosphatidylserine, lysophosphatidylinositol, or lysophosphatidate were relatively ineffective. Lyso-PC-induced arachidonate release was decreased by treatment of cells with PLA2 inhibitors such as para-bromophenacyl bromide and arachidonoyl trifluoromethyl ketone. Furthermore, arachidonate release was attenuated in cells grown in the presence of antisense oligodeoxynucleotides that specifically bind cytosolic PLA2 mRNA. Treatment of cells with lyso-PC resulted in a translocation of PLA2 activity from the cytosolic to the membrane fractions of cells. Lyso-PC induced a rapid influx of Ca2+ from the medium into the cells, with a simultaneous enhancement of protein kinase C (PKC) activity in the membrane fractions. The lyso-PC-induced arachidonate release was attenuated when cells were preincubated with specific inhibitors of PKC (staurosporine and Ro31-8220) or a specific inhibitor of mitogen-activated protein kinase/extracellular regulated kinase kinase (PD098059). Taken together, the results of this study show that lyso-PC caused the elevation of cellular Ca2+ and the activation of PKC, which stimulated cytosolic PLA2 in an indirect manner and resulted in an enhanced release of arachidonate.

Recent evidence suggests that nuclei possess Ca2+ transport mechanisms to regulate nucleoplasmic/cytosolic Ca2+ gradients. We, therefore, investigated the possibility that Ca(2+)-binding proteins may also exist within the nucleus. Electrophoretic analysis revealed the presence of an acidic 93-kDa protein (p93) in the membranes of isolated nuclei. p93 stained blue with "Stains-All" in SDS-polyacrylamide gels and was the major 45Ca(2+)- and ruthenium red-binding nuclear envelope protein in electroblot overlays. p93 was resistant to extraction by 6 M urea but was solubilized in 2% Triton X-100. Citric acid was highly effective in removing the outer nuclear membrane (ER) with concomitant reduction (< 10-fold) of mannose-6-phosphatase activity, but not p93. 45Ca(2+)-binding assays of purified p93 revealed the presence of high capacity Ca(2+)-binding sites comparable to calreticulin. This evidence strongly suggests that p93 is a major Ca(2+)-binding protein of the inner nuclear envelope membrane. Partial amino acid sequence analysis revealed that p93 was close to 100% homologous with a recently identified ER Ca(2+)-binding protein known as calnexin. It is likely, therefore, that p93 is calnexin. However, mild CHAPS detergent treatment of nuclear envelopes and ER revealed distinctly different solubility properties of each membrane for the extraction of p93. This, together with the citrate data, strongly suggests that p93/calnexin, in isolated nuclear envelopes, is mostly bound to the inner membrane. It is possible that p93 may be involved with the regulation of Ca2+ transients between the nucleoplasm and perinuclear space.

The effect of oxidized low density lipoprotein (oxLDL) on the Ca2+ transients of isolated rabbit cardiomyocytes was assessed. LDL was oxidized by a free radical generating system in vitro. The systolic Ca2+ concentration in transients was significantly increased after treatment with 100 micrograms of oxLDL cholesterol/ml for 16 min without having any effect on the diastolic [Ca2+]. However, a toxic effect was observed when the concentration of oxLDL was increased to 1,000 micrograms/ml. Cells treated with 100 micrograms of oxLDL/ml were more sensitive to the blocking action of nicardipine on the Ca2+ transient than were control cardiomyocytes. Furthermore, oxLDL failed to produce a stimulatory effect on the transient when the extracellular [Ca2+] was lowered. The malondialdehyde content in the oxLDL correlated well with the change in systolic [Ca2+] of treated cells. Lazaroid, a novel antioxidant, completely inhibited malondialdehyde formation in the oxLDL and prevented the increment of systolic [Ca2+] in the treated cells. The data indicate that oxLDL can induce relatively rapid alterations in cellular Ca2+ transients via a modification of Ca2+ entry through the L-type Ca2+ channel. LDL oxidation induced by free radicals may play an important role in influencing cardiac contractile function during pathological conditions such as ischemia/reperfusion challenge.

The purpose of this study was to examine the effects of low density lipoprotein (LDL) on Ca2+ transients of isolated rabbit cardiomyocytes. Incubation of cardiomyocytes with > or = 1 mg of LDL cholesterol/ml of perfusion medium induced a slow (> or = 30 min) but significant increase (2-fold) in the cellular Ca2+ transient. The time course for the effect was similar to that observed for the accumulation of cholesterol in the cells. Using Dil- labeled LDL as a fluorescent marker for LDL interaction with the cardiomyocytes, it was concluded that LDL interacted via a receptor-mediated event, but probably this was not the primary mechanism whereby the lipid entered the cell. LDL-treated cells were resistant to the depressant actions for ryanodine, nicardipine, and dichlorobenzamil on the cellular Ca2+ transient. Lowering the extracellular Ca2+ concentration removed the stimulatory effect of LDL on the Ca2+ transient. It is concluded that LDL can induce an increase in the magnitude of the Ca2+ transient in isolated cardiomyocytes. This is a relatively slow process. The mechanism appears to involve a stimulation of a transsarcolemmal Ca2+ transport pathway. These findings have important implications for cardiac contractile function in hypercholesterolemic and drug-treated hypercholesterolemic subjects.

Low molecular weight GTP-binding proteins and their cellular interactions were examined in cardiac muscle. Heart homogenate was separated into various subcellular fractions by differential and sucrose density gradient centrifugation. Various fractions were separated by sodium dodecyl sulfate-gel electrophoresis, blotted to nitrocellulose, and GTP-binding proteins detected by incubating with [alpha-32]GTP. Three polypeptides of M(r) 23,000, 26,000, and 29,000 were specifically labeled with [alpha-32P]GTP in all the fractions examined and enriched in sarcolemmal membranes. The 23-kDa polypeptide was labeled to a higher extent with [alpha-32P]GTP than the 26- and 29-kDa polypeptides. A polypeptide of M(r) 40,000 was weakly labeled with [alpha-32P]GTP in the sarcolemmal membrane and tentatively identified as Gi alpha by immunostaining with anti-Gi alpha antibodies. Cytosolic GTP-binding proteins were labeled with [alpha-32P]GTP and their potential sites of interaction investigated using the blot overlay approach. A polypeptide of 32 kDa present in sarcolemmal membranes, intercalated discs, and enriched in heart gap junctions was identified as a major site of interaction. The low molecular weight GTP-binding proteins associated with the 32-kDa polypeptide through a complex involving cytosolic components of M(r) 56,000, 36,000, 26,000, 23,000, and 12,000. A monoclonal antibody against connexin 32 from liver strongly recognized the 32-kDa polypeptide in heart gap junctions, whereas polyclonal antibodies only weakly reacted with this polypeptide. The low molecular weight GTP-binding proteins associated with a 32-kDa polypeptide in liver membranes that was also immunologically related to connexin 32. These results indicate the presence of a subset of low molecular weight GTP-binding proteins in a membrane-associated and a cytoplasmic pool in cardiac muscle. Their association with a 32-kDa component that is related to the connexins suggests that these polypeptides may be uniquely situated to modulate communication at the cell membrane.

Modification of the cholesterol content of highly purified cardiac sarcolemma from dog ventricles was accomplished by incubation with phosphatidylcholine liposomes containing various amounts of cholesterol. The degree of cholesterol enrichment could be varied by changing the liposomal cholesterol/phospholipid ratio or varying the liposome-membrane incubation time. Na+-Ca2+ exchange measured in cholesterol-enriched sarcolemmal vesicles was increased up to 48% over control values. The stimulation of Na+-Ca2+ exchange was associated with an increased affinity of the exchanger for Ca2+ (Km = 17 microM compared with Km = 22 microM for control preparations). Na+-Ca2+ exchange measured in cholesterol-depleted membrane preparations was decreased by 15%. This depressed activity was associated with a decreased affinity of the exchanger for Ca2+ (Km = 27 microM). These changes were not due to either a change in membrane permeability to Ca2+ or an increase in the amount of Ca2+ bound to sarcolemmal vesicles. The stimulating effect of cholesterol enrichment was specific to the Na+-Ca2+ exchange process since sarcolemmal Ca2+-Mg2+ ATPase activity was depressed 40% by cholesterol enrichment. Further, K+-p-nitrophenylphosphatase and Na+-K+ ATPase activities were depressed in both cholesterol-depleted and cholesterol-enriched sarcolemmal vesicles. In situ oxidation of membrane cholesterol completely eliminated Na+-Ca2+ exchange. These results suggest that cholesterol is intimately associated with Na+-Ca2+ exchange and may interact with the exchange protein and modulate its activity.

Glyceraldehyde-phosphate dehydrogenase and phosphoglycerate kinase activities were associated with a particulate fraction in a cardiac homogenate preparation. NaCl treatment displaced these activities from the particulate fraction. These membrane-associated glycolytic enzymes were gradually lost during the rigorous isolation procedures needed to purify sarcolemmal and sarcoplasmic reticular vesicles. However, exogenously added enzymes did bind to these purified membranes in relatively large quantities (glyceraldehyde-phosphate dehydrogenase: 1.14 mg of enzyme/mg of sarcolemma, 0.30 mg of enzyme/mg of sarcoplasmic reticulum; phosphoglycerate kinase: 1.02 mg of enzyme/mg of sarcolemma, 0.12 mg of enzyme/mg of sarcoplasmic reticulum), which suggests a nonspecific type of binding interaction. Approximately 50% of the glyceraldehyde-phosphate dehydrogenase and 90% of the phosphoglycerate kinase were inactivated upon binding. NaCl removed the enzyme from the membranes in a concentration-dependent manner similar to its effects in the particulate fraction. Dimethonium (5 mM), an organic divalent cation which screens membrane-surface charge, removed greater than 90% glyceraldehyde-phosphate dehydrogenase from the sarcolemmal membrane. These results, and those with NaCl, suggest that the binding of the enzyme to the membrane is charge-related. Phospholipids extracted from the sarcolemmal membrane were capable of binding and inactivating both glycolytic enzymes. Purified anionic phospholipids were more potent at binding and inactivating both enzymes than were neutral phospholipids. These data show that glyceraldehyde-phosphate dehydrogenase and phosphoglycerate kinase can bind to sarcolemmal and sarcoplasmic reticular membranes. This binding is reversible, charge-dependent, and inhibitory. This binding is likely to involve anionic membrane phospholipids.

The purpose of this investigation was to study the effects of a distinct type of phospholipase C on sarcolemmal Na+-Ca2+ exchange. With this phospholipase C (Staphylococcus aureus), treatment of cardiac sarcolemmal vesicles resulted in a specific hydrolysis of membrane phosphatidylinositol. This hydrolysis of phosphatidylinositol also released two proteins (110 and 36 kDa) from the sarcolemmal membrane. Phospholipase C pretreatment of the sarcolemma resulted in an unexpected stimulation of Na+-Ca2+ exchange. The Vmax of Na+-Ca2+ exchange was increased but the Km for Ca2+ was not altered. This stimulation was specific to the Na+-Ca2+ exchange pathway. ATP-dependent Ca2+ uptake was depressed after phospholipase C treatment, but passive membrane permeability to Ca2+ was unaffected. Sarcolemmal Na+,K+-ATPase activity was not altered, whereas passive Ca2+ binding was modestly decreased after phospholipase C pretreatment. The stimulation of Na+-Ca2+ exchange after phosphatidylinositol hydrolysis was greater in inside-out vesicles than in a total population of vesicles of mixed orientation. This finding suggests that the cardiac sarcolemmal Na+-Ca2+ exchanger is functionally asymmetrical. The results also suggest that membrane phosphatidylinositol is inhibitory to the Na+-Ca2+ exchanger or, alternatively, this phospholipid may anchor an endogenous inhibitory protein in the sarcolemmal membrane. The observation that a transsarcolemmal Ca2+ flux pathway may be stimulated solely by phosphatidylinositol hydrolysis independently of phosphoinositide metabolic products like inositol triphosphate is novel.