The vasodilator-stimulated phosphoprotein (VASP) is associated with actin filaments and focal adhesions, which form the interface between the cytoskeleton and the extracellular matrix. VASP is phosphorylated by both the cAMP-and cGMP-dependent protein kinases in a variety of cells, including platelets and smooth muscle cells. Since both the cAMP and cGMP signalling cascades relax smooth muscle and inhibit platelet activation, it was speculated that VASP mediates these effects by modulating actin filament dynamics and integrin activation. To study the physiological relevance of VASP in these processes, we inactivated the VASP gene in mice. Adult VASP-deficient mice had normal agonist-induced contraction, and normal cAMP-and cGMP-dependent relaxation of intestinal and vascular smooth muscle. In contrast, cAMP-and cGMP-mediated inhibition of platelet aggregation was significantly reduced in the absence of VASP. Other cAMP-and cGMPdependent effects in platelets, such as inhibition of agonist-induced increases in cytosolic calcium concentrations and granule secretion, were not dependent on the presence of VASP. Our data show that two different cyclic, nucleotide-dependent mechanisms are operating during platelet activation: a VASP-independent mechanism for inhibition of calcium mobilization and granule release and a VASP-dependent mechanism for inhibition of platelet aggregation which may involve regulation of integrin function.

The generation of nitric oxide (NO) in penile erectile tissue and the subsequent elevation of cyclic GMP (cGMP) levels are important for normal penile erection. Current treatments of erectile dysfunction elevate either cGMP levels by blocking cGMP degrading phosphodiesterase 5 or cyclic AMP (cAMP) levels by intrapenile injection of prostaglandin E1. The molecular target or targets of cGMP in erectile tissue and the role of cAMP for normal penile erection are not known. Herein, we report that mice lacking cGMP-dependent kinase I (cGKI) have a very low ability to reproduce and that their corpora cavernosa fail to relax on activation of the NO͞cGMP signaling cascade. Elevation of cAMP by forskolin, however, induces similar relaxation in normal and cGKI-null corpus cavernosum. In addition, sperm derived from cGKI-null mice is normal, can undergo acrosomal reactions, and can efficiently fertilize eggs. Altogether, these data identify cGKI as the downstream target of cGMP in erectile tissue and provide evidence that cAMP signaling cannot compensate for the absence of the cGMP͞cGKI signaling cascade in vivo.

Nitric oxide (NO)-mediated smooth muscle relaxation is mediated by cGMP through activation of cGMP-dependent protein kinase I (cGKI). We studied the importance of cGKI for lower urinary tract function in mice lacking the gene for cGKI (cGKI-/-) and in litter-matched wild-type mice (cGKI+/+) in vitro and in vivo. cGKI deficiency did not result in any changes in bladder gross morphology or weight. Urethral strips from cGKI-/- mice showed an impaired relaxant response to nerve-derived NO. The cGMP analog 8-bromo-cGMP (8-BrcGMP) and the NO-donor SIN-1 relaxed the wild-type urethra (50-60%) but had only marginal effects in the cGKI-deficient urethra. Bladder strips from cGKI-/- mice responded normally to electrical field stimulation and to carbachol but not to 8-BrcGMP. In vivo, the cGKI-deficient mice showed bladder hyperactivity characterized by decreased intercontraction intervals and nonvoiding bladder contractions. Loss of cGKI abolishes NO-cGMP-dependent relaxations of urethral smooth muscle and results in hyperactive voiding. These data suggest that certain voiding disturbances may be associated with impaired NO-cGKI signaling.

1 Guanosine 3', 5'-cyclic monophosphate (cyclic GMP)-dependent kinase I (cGKI) is a major receptor for cyclic GMP in a variety of cells. Mice lacking cGKI exhibit multiple phenotypes, including severe defects in smooth muscle function. We have investigated the NO/cGMP-and vasoactive intestinal polypeptide (VIP)/adenosine 3', 5'-cyclic monophosphate (cyclic AMP)-signalling pathways in the gastric fundus of wild type and cGKI-de®cient mice. 2 Using immunohistochemistry, similar staining patterns for NO-synthase, cyclic GMP-and VIPimmunoreactivities were found in wild type and cGKI-de®cient mice. 3 In isolated, endothelin-1 (3 nM ± 3 mM)-contracted, muscle strips from wild type mice, electrical ®eld stimulation (1 ± 16 Hz) caused a biphasic relaxation, one initial rapid, followed by a more slowly developing phase. In preparations from cGKI-de®cient mice only the slowly developing relaxation was observed. 4 The responses to the NO donor, SIN-1 (10 nM ± 100 mM), and to 8-Br-cyclic GMP (10 nM ± 100 mM) were markedly impaired in strips from cGKI-de®cient mice, whereas the responses to VIP (0.1 nM ± 1 mM) and forskolin (0.1 nM ± 1 mM) were similar to those in wild type mice. 5 These results suggest that cGKI plays a central role in the NO/cGMP signalling cascade producing relaxation of mouse gastric fundus smooth muscle. Relaxant agents acting via the cyclic AMP-pathway can exert their eects independently of cGKI.

Small nuclear ribonucleoproteins (snRNPs) are particles present only in eukaryotic cells. They are involved in a large variety of RNA maturation processes, most notably in pre-mRNA splicing. Several of the proteins typically found in snRNPs contain a sequence signature, the Sm domain, conserved from yeast to mammals. By using a promoter trap strategy to target actively transcribed loci in murine embryonic stem cells, a new murine gene encoding an Sm motif-containing protein was identified. Database searches revealed that it is the mouse orthologue of Lsm4p, a protein found in yeast and human cells and putatively associated with U6 snRNA. Introduction of the geo reporter gene cassette under the control of the murine Lsm4 (mLsm4) endogenous promoter showed that the gene was ubiquitously transcribed in embryonic and adult tissues. The insertion of the geo cassette disrupted the mLsm4 allele, and homozygosity for the mutation led to a recessive embryonic lethal phenotype. mLsm4-null zygotes survived to the blastocyst stages, implanted into the uterus, but died shortly thereafter. The early death of mLsm4p-null mice suggests that the role of mLsm4p in splicing is essential and cannot be compensated by other Lsm proteins.Spliceosomal U small nuclear ribonucleoproteins (snRNPs) of the Sm class are a group of RNA-protein complexes which are the major components of the spliceosome, the macromolecular complex that catalyzes the pre-mRNA splicing reaction (13,17,18,22). Spliceosomal U snRNPs contain five different snRNAs, U1, U2, U4, U5, and U6. Each is found in different U snRNP particles which display specific functions in splicing. Excluding U6, all snRNAs share a 5Ј-terminal trimethylguanosine (m 3 G) cap and an Sm site. This latter structure consists of a single-stranded U-rich sequence whose consensus is PuA(U n )GPu, where n Ͼ 3. Proteins that bind to individual U snRNAs can be classified into specific and common proteins depending on whether they can associate with one snRNA or with all of them, respectively. At least eight common proteins, termed B, B', D1, D2, D3, E, F, and G, are known so far to associate with all U snRNPs with the exception of U6 snRNP. All these proteins are a major target for autoantibodies in the human disease systemic lupus erythematosus (26) and have a conserved motif named the Sm domain. Sequence comparison shows two Sm consensus sites, termed Sm1 and Sm2 (4, 14, 25), which are linked by a loop-folded spacer and thus form a single protein domain (15). Searches of the sequence databases indicate that the Sm motifs are highly conserved during evolution and are present in proteins (called Sm-like or Lsm) which have no clear counterpart among the eight components of the canonical Sm protein complex (23,25).The precise role of U snRNA binding Sm proteins in splicing reactions is not clear. The only known function of Sm proteins is their crucial role in the biogenesis of U snRNPs. During this process, the nuclear encoded 5Ј-terminal 7-monomethylguanosine-capped (m 7 G) U snRNA is transiently tr...