Functional significance of muscarinic receptor expression within the proximal and distal rat vagina.

Information regarding the role of cholinergic nerves in mediating vaginal smooth muscle contraction is sparse, and in vitro studies of the effects of muscarinic agonists on vaginal smooth muscle are discrepant. The goal of this study was to determine the expression of muscarinic receptors in the vaginal wall of the rat. In addition, we sought to determine the effect of the muscarinic receptor agonist carbachol on contractility and inositol phosphate production of the proximal and distal rat vaginal muscularis. RT-PCR analysis indicated that both M(2) and M(3) receptor transcripts were expressed within the proximal and distal rat vagina. Carbachol dose-dependently (10(-7)-10(-4) M) contracted the rat vaginal muscularis with a greater maximal contractile response in the proximal vagina (P < 0.01) compared with the distal vagina. The contractile responses of the rat vaginal muscularis to carbachol were dose dependently inhibited by the M(3) antagonist para-fluoro-hexahydrosiladefenidol, and a pK(B) of 7.78 and 7.95 was calculated for the proximal and distal vagina, respectively. Inositol phosphate production was significantly increased in both regions of the vagina following 20-min exposure to 50 muM carbachol with higher levels detected in the proximal vagina compared with the distal (P < 0.05). Preliminary experiments indicated the presence of M(2) and M(3) receptors in the human vaginal muscularis as well as contraction of human vaginal muscularis to carbachol, indicating that our animal studies are relevant to human tissue. Our results provide strong evidence for the functional significance of M(3) receptor expression in the vaginal muscularis.

Polyamines, PI(4,5)P2, and actin polymerization.

Spermine (SPM) and spermidine (SPD) activate isolated phosphatidylinositol-4-phosphate 5-kinases (PI(4)P5K), enzymes that convert phosphatidylinositol-4-phosphate to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). PI(4,5)P2 formation is known to be involved in cellular actin reorganization and motility, functions that are also influenced by polyamines. It has not been proven that endogenous polyamines can control inositol phospholipid metabolism. We evoked large decreases in SPD and putrescine (PUT) contents in HL60 cells, using the ornithine decarboxylase inhibitor, alpha-difluoromethylornithine (DFMO), which resulted in decreases in PI(4,5)P2 content per cell and inositol phosphate formation to 76.9 +/- 3.5% and 81.5 +/- 4.0% of control, respectively. Accurately reversing DFMO-evoked decreases in SPD content by incubating cells with exogenous SPD for 20 min rescued these decreases. DFMO treatment and SPD rescues also changed the ratio of total cellular PI(4,5)P2 to PIP suggesting involvement of a SPD-sensitive PI(4)P5K. PUT and SPM were not involved in DFMO-evoked changes in cellular PI(4,5)P2 contents. In DFMO-treated HL60 cells, the percent of total actin content that was filamentous was decreased to 59.1 +/- 5.8% of that measured in paired control HL60 cells, a finding that was rescued following reversal of DFMO-evoked decreases in SPD and PI(4,5)P2 contents. In slowly proliferating DMSO-differentiated HL60 cells, inositol phospholipid metabolism was uncoupled from SPD control. We conclude: in rapidly proliferating HL60 cells, but not in slowly proliferating differentiated HL60 cells, there are endogenous SPD-sensitive PI(4,5)P2 pools, probably formed via SPD-sensitive PI(4)P5K, that likely control actin polymerization.

A membrane defect in the pathogenesis of the Smith-Lemli-Opitz syndrome.

The Smith-Lemli-Opitz syndrome (SLOS) is an often lethal birth defect resulting from mutations in the gene responsible for the synthesis of the enzyme 3beta-hydroxy-steroid-Delta7-reductase, which catalyzes the reduction of the double bond at carbon 7 on 7-dehydrocholesterol (7-DHC) to form unesterified cholesterol. We hypothesize that the deficiency in cholesterol biosynthesis and subsequent accumulation of 7-DHC in the cell membrane leads to defective composition, organization, dynamics, and function of the cell membrane. Using skin fibroblasts obtained from SLOS patients, we demonstrate that the SLOS membrane has increased 7-DHC and reduced cholesterol content and abnormal membrane fluidity. X-ray diffraction analyses of synthetic membranes prepared to mimic SLOS membranes revealed atypical membrane organization. In addition, calcium permeability is markedly augmented, whereas membrane-bound Na+/K+ATPase activity, folate uptake, inositol-1,4,5-trisphosphate signaling, and cell proliferation rates are markedly suppressed. These data indicate that the disturbance in membrane sterol content in SLOS, likely at the level of membrane caveolae, directly contributes to the widespread tissue abnormalities in this disease.

Mechanisms of hypotonicity-induced calcium signaling and integrin activation by arachidonic acid-derived inflammatory mediators in B cells.

We previously characterized the initial steps in the activation of novel (calcium-permeant) nonselective cation channels (NSCCs) and calcium release-activated calcium channels in primary murine B lymphocytes. Phospholipase C products, namely diacylglycerol and d-myo-inositol 1,4,5-trisphosphate, were identified as proximal intracellular agonists of these respective channels following mechanical stimulation of B cells. However, neither the distal steps in NSCC activation nor the contribution of these channels to sustained mechanical signaling were defined in these previous studies. In this study, single cell measurements of intracellular Ca(2+) were used to define the mechanisms of NSCC activation and demonstrate a requirement for arachidonic acid liberated from diacylglycerol. Several arachidonic acid-derived derivatives were identified that trigger Ca(2+) entry into B cells, including the lipoxygenase product 5-hydroperoxyeicosatetranenoic acid and the cytochrome P450 hydroxylase product 20-hydroxyeicosatetraenoic; however, the cytochrome P450 epoxygenase product 5,6-epoxyeicosatrienoic acid is primarily responsible for hypotonicity-induced responses. In addition to regulating calcium entry, our data suggest that eicosanoid-activated NSCCs have a separate and direct role in regulating the avidity of integrins on B cells for extracellular matrix proteins, including ICAM-1 and VCAM-1. Thus, in addition to defining a novel osmotically activated signal transduction pathway in B cells, our results have broad implications for understanding how inflammatory mediators dynamically and rapidly regulate B cell adhesion and trafficking.

Nitric oxide stimulated vascular smooth muscle cells undergo apoptosis induced in part by arachidonic acid derived eicosanoids.

The role of eicosanoids in atherogenesis has not been thoroughly explained. This is partly due to the numerous eicosanoids and the variable effects that each has on different systems. Apoptosis of vascular smooth muscle cells has been shown to play a role in the atherosclerotic disease leading to lesion formation and further destabilization of the formed lesion. In this study, we have investigated the role of arachidonic acid derived eicosanoids in nitric oxide (NO)-stimulated vascular smooth muscle cells. We have shown previously that the nitric oxide (NO)-induced apoptosis of vascular smooth muscle cells was accompanied by arachidonic acid release via cytoplasmic phospholipase A(2) (cPLA(2)) activation. Also, arachidonic acid, but not oleic acid, induced apoptosis of these cells at low concentrations (5-10 microM). Our results revealed that the cPLA(2) specific inhibitor, arachidonyl trifluoromethyl ketone (AACOCF(3)), blocked NO-induced eicosanoid production, while the presence of arachidonic acid enhanced the ability of the cells to make prostaglandin E(2) (PGE(2)). Also, inhibitors of the cyclo-oxygenase (Cox) enzymes, such as N-[2-cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS-398), a specific Cox-2 inhibitor, or indomethacin, a non-specific Cox inhibitor, blocked NO-induced PGE(2) production and apoptosis of vascular smooth muscle cells to the same extent, indicating that apoptosis might be induced by a Cox-2 metabolic product. In addition to these observations, the eicosanoids investigated, namely, PGE(2), PGI(2) LTB(4), and PGJ(2), showed different effects on vascular smooth muscle cells. Both PGJ(2) and LTB(4) decreased the percentage of viable cells and induced apoptosis of vascular smooth muscle cells, while PGE(2) and PGI(2) had no effect on cell viability and failed to induce apoptosis. These data suggest that eicosanoids, such as PGJ(2), but not PGE(2) or PGI(2), are involved in NO-induced apoptosis of vascular smooth muscle cells and that the eicosanoid synthesis pathways might be utilized for vascular therapeutic strategies.

NO induced apoptosis of vascular smooth muscle cells accompanied by ceramide increase.

We have shown previously that nitric-oxide (NO) can induce apoptosis of vascular smooth muscle cells (VSMCs) and that the NO-induced apoptosis is accompanied by an increase in arachidonic acid release via cytoplasmic Ca(2+)-dependent phospholipase A(2) (cPLA(2)). We have evidence that during NO-induced apoptosis there is an increase in ceramide synthesis. The use of inhibitors of ceramide synthesis, namely, fumonisin B1 and desipramine, which block ceramide synthase and sphingomyelinase, respectively revealed that the ceramide was produced via the sphingomyelinase pathway. Inhibition of acid sphingomyelinase by desipramine was shown to inhibit NO-induced apoptosis while fumonisin B1 failed to inhibit this process. C(2)-ceramide could induce apoptosis in cultured VSMCs. Apoptosis in smooth muscle cells was accompanied by the increased activity of DNA fragmentation factor-40 and the secretion of cathepsin D from the cells. In this study, ceramide appears to function as a mediator of apoptosis.

cPLA2 activator peptide, PLAP, increases arachidonic acid release and apoptosis of vascular smooth muscle cells.

Apoptosis of vascular smooth muscle cells (VSMCs) has recently drawn a lot of interest in various laboratories due to its importance in atherogenesis. We have shown previously that nitric-oxide (NO) can induce apoptosis of VSMCs and that the NO-induced apoptosis is accompanied by an increase in arachidonic acid release via cytoplasmic Ca(2+)-dependent phospholipase A(2) (cPLA(2)). We have demonstrated here that NO-induced activation of cPLA(2) leading to increased arachidonic acid release can be mimicked via direct activation of cPLA(2) with a cPLA(2) activator peptide, PLAP. The PLAP induced arachidonic acid release and apoptosis is inhibitable by a cPLA(2)-specific inhibitor, AACOCF(3), indicating the direct involvement of cPLA(2). In this study, activation of cPLA(2) appears to be preceded by activation and binding by PLAP indicating that the cPLA(2) functions are mediated via PLAP.

Subcellular localization of phospholipase C isoforms in vascular smooth muscle.

The phospholipase C (PLC) isoform most important during agonist-activated IP(3) production in vascular smooth muscle is still unknown. When PLC activity in rat tail artery homogenate was determined, this activity was shown to be inhibited by an antibody directed against PLCbeta2. Antibodies directed against the gamma1, beta1, beta3 and delta1 isoforms of PLC failed to inhibit PLC activity in this tissue. Both PLCbeta2 and PLCgamma1 were isolated from rat tail artery by DEAE column chromatography and PLCbeta2 activity was shown to be 3-fold greater than PLCgamma1 activity. When rat tail artery was treated with norepinephrine (10 mM), PLCbeta2 was shown to translocate from cytosol to membranes. When subcellular fractions of rat tail artery were isolated by sucrose density gradient centrifugation, including nuclei, plasma membrane, and cytosol, PLCbeta2 was detected in the plasma membrane and the cytosol but not in the nuclei. PLCdelta1 and PLCgamma1 were found only in cytosol. This evidence is consistent with the model wherein an agonist such as norepinephrine can activate smooth muscle contraction via interaction with a plasma membrane receptor which can easily interact with a plasma membrane-associated isoform of PLC, such as PLCbeta2.

Arachidonic acid release by cPLA2 may be causally related to NO-induced apoptosis in vascular smooth muscle cells.

Apoptosis has been shown to occur in vascular smooth muscle cells during the development of atherosclerosis. In order to investigate the possible role of arachidonic acid during apoptosis in vascular smooth muscle, we induced apoptosis in cultured rat aortal smooth muscle cells (SMCs) by treatment with either UV (ultraviolet) radiation, tumor necrosis factor-alpha (TNF-alpha) or NO donor drugs (sodium nitroprusside, or S-nitroso-N-acetyl-D-penicillamine, SNAP). Apoptosis was detected by either DNA fragmentation analysis or by TUNEL analysis. UV radiation, TNF-alpha and NO were observed to stimulate apoptosis in the cells as well as to stimulate arachidonate release from the cells. NO also increased levels of cPLA2 in the cells, which is an enzyme that is frequently activated in cells that release arachidonate. These agents stimulated arachidonate release somewhat earlier than they stimulated apoptosis in the cells. The inhibition of cPLA2 by arachidonyl trifluoromethyl ketone (AACOCF3) also led to the inhibition of arachidonate release from the cells as well as the inhibition of nitroprusside stimulated apoptosis. Arachidonic acid itself could induce apoptosis in the cultured cells. These observations provide evidence that arachidonate may be involved in apoptosis in vascular smooth muscle.