Rescue of cardiac α-actin-deficient mice by enteric smooth muscle γ-actin

The muscle actins in higher vertebrates display highly conserved amino acid sequences, yet they show distinct expression patterns. Thus, cardiac α-actin, skeletal α-actin, vascular smooth muscle α-actin, and enteric smooth muscle γ-actin comprise the major actins in their respective tissues. To assess the functional and developmental significance of cardiac α-actin, the murine (129/SvJ) cardiac α-actin gene was disrupted by homologous recombination. The majority (≈56%) of the mice lacking cardiac α-actin do not survive to term, and the remainder generally die within 2 weeks of birth. Increased expression of vascular smooth muscle and skeletal α-actins is observed in the hearts of newborn homozygous mutants and also heterozygotes but apparently is insufficient to maintain myofibrillar integrity in the homozygous mutants. Mice lacking cardiac α-actin can be rescued to adulthood by the ectopic expression of enteric smooth muscle γ-actin using the cardiac α-myosin heavy chain promoter. However, the hearts of such rescued cardiac α-actin-deficient mice are extremely hypodynamic, considerably enlarged, and hypertrophied. Furthermore, the transgenically expressed enteric smooth muscle γ-actin reduces cardiac contractility in wild-type and heterozygous mice. These results demonstrate that alterations in actin composition in the fetal and adult heart are associated with severe structural and functional perturbations.     

Muscarinic stimulation of synaptic activity by protein kinase C is inhibited by adenosine in cultured hippocampal neurons

We have studied the effect of the cholinergic agonist carbachol on the spontaneous release of glutamate in cultured rat hippocampal cells. Spontaneous excitatory postsynaptic currents (sEPSCs) through glutamatergic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type channels were recorded by means of the patch-clamp technique. Carbachol increased the frequency of sEPSCs in a concentration-dependent manner. The kinetic properties of the sEPSCs and the amplitude distribution histograms were not affected by carbachol, arguing for a presynaptic site of action. This was confirmed by measuring the turnover of the synaptic vesicular pool by means of the fluorescent dye FM 1–43. The carbachol-induced increase in sEPSC frequency was not mimicked by nicotine, but could be blocked by atropine or by pirenzepine, a muscarinic cholinergic receptor subtype M1 antagonist. Intracellular Ca²⁺ signals recorded with the fluorescent probe Fluo-3 indicated that carbachol transiently increased intracellular Ca²⁺ concentration. Since, however, carbachol still enhanced the sEPSC frequency in bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetate-loaded cells, this effect could not be attributed to the rise in intracellular Ca²⁺ concentration. On the other hand, the protein kinase inhibitor staurosporine as well as a down-regulation of protein kinase C by prolonged treatment of the cells with 4β-phorbol 12-myristate 13-acetate inhibited the carbachol effect. This argues for an involvement of protein kinase C in presynaptic regulation of spontaneous glutamate release. Adenosine, which inhibits synaptic transmission, suppressed the carbachol-induced stimulation of sEPSCs by a G protein-dependent mechanism activated by presynaptic A1-receptors.     

Nonmuscle myosin II-B is required for normal development of the mouse heart

We used targeted gene disruption in mice to ablate nonmuscle myosin heavy chain B (NMHC-B), one of the two isoforms of nonmuscle myosin II present in all vertebrate cells. Approximately 65% of the NMHC-B^−/− embryos died prior to birth, and those that were born suffered from congestive heart failure and died during the first day. No abnormalities were detected in NMHC-B^+/− mice. The absence of NMHC-B resulted in a significant increase in the transverse diameters of the cardiac myocytes from 7.8 ± 1.8 μm (right ventricle) and 7.8 ± 1.3 μm (left ventricle) in NMHC-B^+/+ and B^+/− mice to 14.7 ± 1.1 μm and 13.8 ± 2.3 μm, respectively, in NMHC-B^−/− mice (in both cases, P < 0.001). The increase in size of the cardiac myocytes was seen as early as embryonic day 12.5 (4.5 ± 0.2 μm for NMHC-B^+/+ and B^+/− vs. 7.2 ± 0.6 μm for NMHC-B^−/− mice (P < 0.01)). Six of seven NMHC-B^−/− newborn mice analyzed by serial sectioning also showed structural cardiac defects, including a ventricular septal defect, an aortic root that either straddled the defect or originated from the right ventricle, and muscular obstruction to right ventricular outflow. Some of the hearts of NMHC-B^−/− mice showed evidence for up-regulation of NMHC-A protein. These studies suggest that nonmuscle myosin II-B is required for normal cardiac myocyte development and that its absence results in structural defects resembling, in part, two common human congenital heart diseases, tetralogy of Fallot and double outlet right ventricle.     

The early phase of engraftment after murine blood cell transplantation is mediated by hematopoietic stem cells

Blood cell transplantation is largely replacing bone marrow transplantation because engraftment is more rapid. This accelerated engraftment is thought to be mediated by relatively mature committed hematopoietic progenitor cells. Herein, we have used a modified rhodamine (Rho) staining procedure to identify and purify Rho^+/++ (dull/bright) and Rho⁻ (negative) subpopulations of hematopoietic progenitor cells in murine cytokine-mobilized blood. The Rho^+/++ cell population contained >99% of committed progenitor cells with in vitro colony-forming ability. The Rho⁻ cell population contained the majority of hematopoietic stem cells with in vivo marrow repopulating ability. The rate of hematopoietic reconstitution was identical in recipients of grafts containing only purified Rho⁻ stem cells or purified Rho⁻ stem cells in combination with large numbers of Rho^+/++ committed progenitor cells. In contrast, transplantation of 3-fold more hematopoietic stem cells resulted in accelerated reconstitution, indicating that the reconstitution rate was determined by the absolute numbers of Rho⁻ stem cells in the graft. In addition, we observed a 5- to 8-fold reduced frequency of the subset of hematopoietic stem cells with long-term repopulating ability in cytokine-mobilized blood in comparison to steady-state bone marrow. Our results indicate that hematopoietic stem cells and not committed progenitor cells mediate early hematopoietic reconstitution after blood cell transplantation and that relative to bone marrow, the frequency of stem cells with long-term repopulating ability is reduced in mobilized blood.     

Inactivation of calcium-activated chloride channels in smooth muscle by calcium/calmodulin-dependent protein kinase

To determine the mechanisms responsible for the termination of Ca²⁺-activated Cl⁻ currents (I_Cl(Ca)), simultaneous measurements of whole cell currents and intracellular Ca²⁺ concentration ([Ca²⁺]_i) were made in equine tracheal myocytes. In nondialyzed cells, or cells dialyzed with 1 mM ATP, I_Cl(Ca) decayed before the [Ca²⁺]_i decline, whereas the calcium-activated potassium current decayed at the same rate as [Ca²⁺]_i. Substitution of AMP-PNP or ADP for ATP markedly prolonged the decay of I_Cl(Ca), resulting in a rate of current decay similar to that of the fall in [Ca²⁺]_i. In the presence of ATP, dialysis of the calmodulin antagonist W7, the Ca²⁺/calmodulin-dependent kinase II (CaMKII) inhibitor KN93, or a CaMKII-specific peptide inhibitor the rate of I_Cl(Ca) decay was slowed and matched the [Ca²⁺]_i decline, whereas H7, a nonspecific kinase inhibitor with low affinity for CaMKII, was without effect. When a sustained increase in [Ca²⁺]_i was produced in ATP dialyzed cells, the current decayed completely, whereas in cells loaded with 5′-adenylylimidodiphosphate (AMP-PNP), KN93, or the CaMKII inhibitory peptide, I_Cl(Ca) did not decay. Slowly decaying currents were repeatedly evoked in ADP- or AMP-PNP-loaded cells, but dialysis of adenosine 5′-O-(3-thiotriphosphate) or okadaic acid resulted in a smaller initial I_Cl(Ca), and little or no current (despite a normal [Ca²⁺]_i transient) with a second stimulation. These data indicate that CaMKII phosphorylation results in the inactivation of calcium-activated chloride channels, and that transition from the inactivated state to the closed state requires protein dephosphorylation.     

Mitogenic activity of endothelin-1 and -3 on vascular smooth muscle cells is inhibited by atrial natriuretic peptides

The effect of endothelin-1 (ET-1) and endothelin-3 (ET-3) on the proliferation of aortic vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) was assessed by the measurement of (3H)-thymidine incorporation into DNA. ET-1 and ET-3 stimulated the DNA synthesis in a dose-dependent manner. ET-3 was less potent than ET-1 in its stimulatory activity. The increase in (3H)-thymidine incorporation is clearly attenuated by Atriopeptin III (ANP 5-28). Atriopeptin I (ANP 5-25), a ANP fragment with the reduced biological activity compared to ANP 5-28, has no effect on the endothelin mediated stimulation of DNA-synthesis.     

Proliferation of smooth muscle cells after vascular injury is inhibited by an antibody against basic fibroblast growth factor.

Proliferation of smooth muscle cells (SMCs) represents an important event in vascular lesion formation. Despite the common belief that growth factors contribute to the development of the atherosclerotic plaque, until now there has been no direct evidence for a role of mitogens in the development of arterial lesions. Balloon catheter injury of the rat carotid artery is accompanied by death of medial SMCs and is typically followed by proliferation of SMCs with subsequent formation of an intimal lesion. Our hypothesis is that injury causes mitogens to be released from dead cells, which then stimulate cell proliferation. One such mitogen that may be important in this process is basic fibroblast growth factor (bFGF), which can be detected immunocytochemically in SMCs and endothelial cells of adult rat carotid arteries. Systemic injection of a neutralizing antibody against bFGF prior to balloon catheterization significantly decreased the induced SMC proliferation by approximately 80%. The intimal lesion that developed within 8 days after injury, however, was not significantly reduced. The results of this study support the concept that endogenous bFGF is the major mitogen controlling the growth of vascular smooth muscle cells following injury. These data may have implications for the observed failure of endarterectomy and angioplasty procedures.     

The visual response of retinal ganglion cells is not altered by optic nerve transection in transgenic mice overexpressing Bcl-2

Attempts to rescue retinal ganglion cells from retrograde degeneration have had limited success, and the residual function of surviving neurons is not known. Recently, it has been found that axotomized retinal ganglion cells die by apoptotic mechanisms. We have used adult transgenic mice overexpressing the Bcl-2 protein, a powerful inhibitor of apoptosis, as a model for preventing injury-induced cell death in vivo. Several months after axotomy, the majority of retinal ganglion cells survived and exhibited normal visual responses. In control wild-type mice, the vast majority of axotomized retinal ganglion cells degenerated, and the physiological responses were abolished. These results suggest that strategies aimed at increasing Bcl-2 expression, or mimicking its function, might effectively counteract trauma-induced cell death in the central nervous system. Neuronal survival is a necessary condition in the challenge for promoting regeneration and eventually restoring neuronal function.     

Coatomer, but not P200/myosin II, is required for the in vitro formation of trans-Golgi network-derived vesicles containing the envelope glycoprotein of vesicular stomatitis virus

Using a cytosol and nucleotide dependent assay that we previously developed, we have investigated the requirement for coat proteins in the in vitro production of trans-Golgi network (TGN)-derived vesicles from a Madin-Darby canine kidney (MDCK) cell Golgi fraction that contains the ³⁵S-labeled, terminally glycosylated, envelope glycoprotein of vesicular stomatitis virus (VSV-G) accumulated in the TGN. We found that the TGN-derived vesicles, like those involved in intra-Golgi transport and in retrograde transport to the endoplasmic reticulum, contain a coatomer coat and that coatomer is required for their formation. Thus, after they are produced with GTPγS, the coated vesicles could be captured on beads containing anticoatomer antibody. Moreover, a cytosolic protein fraction depleted of coatomer could not support vesicle formation but it did so after purified coatomer was added. We also determined that P200/myosin II does not play an essential role in the in vitro generation of TGN-derived vesicles. Thus, removal of this protein from the cytosol, by differential salt precipitation or binding to phalloidin-induced actin filaments, had no effect on vesicle generation. Nevertheless, immunodepletion of cytosol using the anti-P200/myosin II AD7 antibody abolished vesicle generation and that antibody was capable of effectively immunocapturing coated vesicles, even when these were generated in the absence of P200/myosin II. These effects, however, are explained by the unexpected finding that the AD7 antibody interacts with undenatured coatomer.     

The motor domain and the regulatory domain of myosin solely dictate enzymatic activity and phosphorylation-dependent regulation, respectively

While the structures of skeletal and smooth muscle myosins are homologous, they differ functionally from each other in several respects, i.e., motor activities and regulation. To investigate the molecular basis for these differences, we have produced a skeletal/smooth chimeric myosin molecule and analyzed the motor activities and regulation of this myosin. The produced chimeric myosin is composed of the globular motor domain of skeletal muscle myosin (Met¹–Gly⁷⁷³) and the C-terminal long α-helix domain of myosin subfragment 1 as well as myosin subfragment 2 (Gly⁷⁷³–Ser¹¹⁰⁴) and light chains of smooth muscle myosin. Both the actin-activated ATPase activity and the actin-translocating activity of the chimeric myosin were completely regulated by light chain phosphorylation. On the other hand, the maximum actin-activated ATPase activity of the chimeric myosin was the same as skeletal myosin and thus much higher than smooth myosin. These results show that the C-terminal light chain-associated domain of myosin head solely confers regulation by light chain phosphorylation, whereas the motor domain determines the rate of ATP hydrolysis. This is the first report, to our knowledge, that directly determines the function of the two structurally separated domains in myosin head.     

Activation and inhibition of K-ATP currents by guanine nucleotides is mediated by different channel subunits

The ATP-sensitive potassium channel (K-ATP channel) plays a key role in insulin secretion from pancreatic β-cells. It is closed by glucose metabolism, which stimulates secretion, and opened by the drug diazoxide, which inhibits insulin release. Metabolic regulation is mediated by changes in ATP and MgADP concentration, which inhibit and potentiate channel activity, respectively. The β-cell K-ATP channel consists of a pore-forming subunit, Kir6.2, and a regulatory subunit, SUR1. The site at which ATP mediates channel inhibition lies on Kir6.2, while the potentiatory action of MgADP involves the nucleotide-binding domains of SUR1. K-ATP channels are also activated by MgGTP and MgGDP. Furthermore, both nucleotides support the stimulatory actions of diazoxide. It is not known, however, whether guanine nucleotides mediate their effects by direct interaction with one or more of the K-ATP channel subunits or indirectly via a GTP-binding protein. We used a truncated form of Kir6.2, which expresses independently of SUR1, to show that GTP blocks K-ATP currents by interaction with Kir6.2 and that the potentiatory effects of GTP are endowed by SUR1. We also showed that mutation of the lysine residue in the Walker A motif of either the first (K719A) or second (K1384M) nucleotide-binding domain of SUR1 abolished both the potentiatory effects of GTP and GDP on K-ATP currents and their ability to support stimulation by diazoxide. This argues that the stimulatory effects of guanine nucleotides require the presence of both Walker A lysines.     

Activation of mitogen-activating protein kinase by glucose is not required for insulin secretion

In the insulinoma cell line INS-1, a model system for glucose-regulated insulin secretion, the mitogen-activating protein (MAP) kinases/extracellular signal-regulated protein kinases, ERK1 and ERK2 are activated up to 15-fold by physiological concentrations of glucose, in the range of 3–12 mM. The related MAP kinase family members, the c-Jun–N-terminal kinases/stress-activated protein kinases are insensitive to glucose, while the p38 MAP kinase is slightly glucose responsive (1.5-fold). ERK activation is dependent on glucose metabolism and the subsequent increase in calcium influx. Inhibiting activation of ERK1 and ERK2 with the MEK1/2 inhibitor PD98059 has no effect on insulin secretion, indicating that ERK activity is not necessary for secretion under these conditions. Glucose activates ERK1 and ERK2 in cytosolic and purified nuclear fractions of INS-1 cells and more of each is found in nuclei from glucose-treated cells. These findings suggest that some of the glucose-dependent actions of ERKs will be exerted in the nucleus.     

Inhibition of tumorigenesis by a cytosine–DNA, methyltransferase, antisense oligodeoxynucleotide

This paper tests the hypothesis that cytosine DNA methyltransferase (DNA MeTase) is a candidate target for anticancer therapy. Several observations have suggested recently that hyperactivation of DNA MeTase plays a critical role in initiation and progression of cancer and that its up-regulation is a component of the Ras oncogenic signaling pathway. We show that a phosphorothioate-modified, antisense oligodeoxynucleotide directed against the DNA MeTase mRNA reduces the level of DNA MeTase mRNA, inhibits DNA MeTase activity, and inhibits anchorage independent growth of Y1 adrenocortical carcinoma cells ex vivo in a dose-dependent manner. Injection of DNA MeTase antisense oligodeoxynucleotides i.p. inhibits the growth of Y1 tumors in syngeneic LAF1 mice, reduces the level of DNA MeTase, and induces demethylation of the adrenocortical-specific gene C21 and its expression in tumors in vivo. These results support the hypothesis that an increase in DNA MeTase activity is critical for tumorigenesis and is reversible by pharmacological inhibition of DNA MeTase.     

Serum-induced proliferation of rabbit aortic smooth muscle cells from the contractile state is inhibited by 8-Br-cAMP but not 8-Br-cGMP

A method is described for the quantification of vascular smooth muscle cell growth from individual explants of contractile rabbit aortic tunica media. The precision of the method probably depends on regular explant geometry (1-mm squares) and pooling sufficient explants. Serum-induced growth was quantified by measurements of ATP concentration, incorporation of [3H]thymidine and DNA concentration. The possible effects of endogenous vasodilator agents on growth were investigated by using lipid soluble analogues of their second messengers, namely 8-Br-cAMP and 8-Br-cGMP, which are known to relax rabbit aortic strips. Cell growth was inhibited concentration-dependently by 8-Br-cAMP but not 8-Br-cGMP (0.01-1 mM). The effect of 8-Br-cAMP was reversible, and also occurred when addition was delayed until after growth had commenced. The results imply that endogenous vasodilators such as prostacyclin, adenosine and adrenaline, which increase cAMP concentration, may normally suppress smooth muscle cell growth, whereas nitric oxide and atriopeptins, which increase cGMP concentration, may not.     

Cloning and characterization of TDD5, an androgen target gene that is differentially repressed by testosterone and dihydrotestosterone

By using mRNA polymerase chain reaction differential display technique (DDPCR), we have identified one early responsive cDNA fragment, TDD5, from a 5α-reductase-deficient T cell hybridoma. The DDPCR profiles of TDD5 suggest that its expression can be repressed by testosterone (T) within 2 hr. More importantly, both DDPCR and Northern blot analysis further demonstrated that the expression of TDD5 was differentially repressed by T and dihydrotestosterone (DHT) at the mRNA level. To our knowledge, this is the first androgen target gene to show a preference in response to T over DHT in cell culture. TDD5 is expressed in several tissues with particular abundance in kidney. Full-length TDD5 cDNA (2,916 bp) encodes a protein with a calculated molecular weight of 42,000. Finally, our animal studies further confirm that TDD5 mRNA levels can be repressed to the basal level 8 hr after DHT administration. The isolation and characterization of the early-responsive androgen target gene TDD5 and the fact that TDD5 mRNA level can be differentially regulated by T and DHT may provide a useful tool to study the molecular mechanism of androgen preference on target gene regulation.     

Independent, specialized invasions of ectomycorrhizal mutualism by two nonphotosynthetic orchids

We have investigated the mycorrhizal associations of two nonphotosynthetic orchids from distant tribes within the Orchidaceae. The two orchids were found to associate exclusively with two distinct clades of ectomycorrhizal basidiomycetous fungi over wide geographic ranges. Yet both orchids retained the internal mycorrhizal structure typical of photosynthetic orchids that do not associate with ectomycorrhizal fungi. Restriction fragment length polymorphism and sequence analysis of two ribosomal regions along with fungal isolation provided congruent, independent evidence for the identities of the fungal symbionts. All 14 fungal entities that were associated with the orchid Cephalanthera austinae belonged to a clade within the Thelephoraceae, and all 18 fungal entities that were associated with the orchid Corallorhiza maculata fell within the Russulaceae. Restriction fragment length polymorphism and single-strand conformational polymorphism analysis of ectomycorrhizal tree roots collected adjacent to Cephalanthera showed that (i) the fungi associated internally with Cephalanthera also form typical external ectomycorrhizae and that (ii) ectomycorrhizae formed by other Basidiomycetes were abundant where the orchid grows but these fungi did not associate with the orchid. This is the first proof of ectomycorrhizal epiparasitism in nature by an orchid. We argue that these orchids are cheaters because they do not provide fixed carbon to associated fungi. This view suggests that mycorrhizae, like other ancient mutualisms, are susceptible to cheating. The extreme specificity in these orchids relative to other ectomycorrhizal plants agrees with trends seen in more conventional parasites.     

Functional CD40 ligand is expressed on human vascular endothelial cells, smooth muscle cells, and macrophages: Implications for CD40–CD40 ligand signaling in atherosclerosis

Increasing evidence supports involvement of inflammation and immunity in atherogenesis. We report here that CD40 ligand (CD40L), an immunoregulatory signaling molecule heretofore considered largely restricted to recently activated CD4⁺ T lymphocytes, is expressed by human vascular endothelial cells (EC), smooth muscle cells (SMC), and human macrophages in vitro, and is coexpressed with its receptor CD40 on all three cells types in human atherosclerotic lesions in situ. Cultured human vascular EC, SMC, and human macrophages all constitutively expressed CD40L mRNA as well as protein. Stimulation with interleukin 1β, tumor necrosis factor α, or interferon γ increased surface levels and de novo synthesis of CD40L on all three cell types. CD40L expressed on EC, SMC, and macrophages exhibited biological activity, as it induced B7.2 expression on B cells. Human vascular SMC also constitutively expressed CD40, the receptor for CD40L, and through CD40 signaling, human recombinant CD40L induced expression of proinflammatory cytokines in these cells, identifying SMC as a target for CD40L. Human atherosclerotic lesions (n = 8) showed expression of immunoreactive CD40L on EC, SMC, and macrophages, while normal arterial tissues (n = 5) contained no CD40L. In atheroma CD40L⁺ cells often also expressed CD40. These observations establish human vascular EC, SMC, and human macrophages as a novel source of CD40L, and point to T cell-independent CD40 signaling, and a broader function of this pathway in regulation of nonimmune cells, as illustrated here by potential autocrine and paracrine activation during atherogenesis.