Biomedical subjects

K W Cho

Publications and source records attributed to K W Cho.

At least 37 records · Page 2Linked to original sources

PubMed2000

Xlim-1 and LIM domain binding protein 1 cooperate with various transcription factors in the regulation of the goosecoid promoter.

The homeobox genes Xlim-1 and goosecoid (gsc) are coexpressed in the Spemann organizer and later in the prechordal plate that acts as head organizer. Based on our previous finding that gsc is a possible target gene for Xlim-1, we studied the regulation of gsc transcription by Xlim-1 and other regulatory genes expressed at gastrula stages, by using gsc-luciferase reporter constructs injected into animal explants. A 492-bp upstream region of the gsc promoter responds to Xlim-1/3m, an activated form of Xlim-1, and to a combination of wild-type Xlim-1 and Ldb1, a LIM domain binding protein, supporting the view that gsc is a direct target of Xlim-1. Footprint and electrophoretic mobility shift assays with GST-homeodomain fusion proteins and embryo extracts overexpressing FLAG-tagged full-length proteins showed that the Xlim-1 homeodomain or Xlim-1/Ldb1 complex recognize several TAATXY core elements in the 492-bp upstream region, where XY is TA, TG, CA, or GG. Some of these elements are also bound by the ventral factor PV.1, whereas a TAATCT element did not bind Xlim-1 or PV.1 but did bind the anterior factors Otx2 and Gsc. These proteins modulate the activity of the gsc reporter in animal caps: Otx2 activates the reporter synergistically with Xlim-1 plus Ldb1, whereas Gsc and PV.1 strongly repress reporter activity. We show further, using animal cap assays, that the endogenous gsc gene was synergistically activated by Xlim-1, Ldb1, and Otx2 and that the endogenous otx2 gene was activated by Xlim-1/3m, and this activation was suppressed by the posterior factor Xbra. Based on these data, we propose a model for gene interactions in the specification of dorsoventral and anteroposterior differences in the mesoderm during gastrulation.

Animals↗

PubMed2000

Is chordin a long-range- or short-range-acting factor? Roles for BMP1-related metalloproteases in chordin and BMP4 autofeedback loop regulation.

Diffusible morphogen models have been used widely to explain regional specification of tissues and body axes during animal development. The three-signal model for patterning the dorsal-ventral axis of the amphibian embryo proposes, in part, that a factor(s) secreted from Spemann's organizer is responsible for converting lateral marginal zone into more dorsal cell fates. We examine the possibility that chordin, a secreted inhibitor of bone morphogenetic protein (BMP) signaling and candidate "dorsalizing signal," is a long-range-acting factor. We show that chordin can, when overexpressed, act directly over distances of at least 450 microm in the early Xenopus embryo to create a gradient of BMP signaling. However, since lower levels of chordin can still induce secondary axes and these amounts of chordin act only locally to inhibit a BMP target gene, we suggest that chordin likely acts as a short-range signal in vivo. Furthermore, BMP1, a secreted metalloprotease that cleaves chordin protein in vitro, inhibits chordin's axis-inducing effects, suggesting that BMP1 functions to negatively regulate chordin's action in vivo. A dominant-negative mutant BMP1 blocks the in vitro cleavage of chordin protein by wild-type BMP1 and induces secondary axes when injected ventrally. We argue that BMP1 and Xolloid are probably functionally redundant metalloproteases and may have two roles in the early Xenopus embryo. One role may be to inhibit the action of low-level chordin protein expressed throughout the entire embryo and a possible second role may be to inhibit activation of a juxtacrine cell relay, thereby confining chordin's action to the organizer region preventing chordin from functioning as a long-range-acting factor.

Animals↗

PubMed2000

Dishevelled phosphorylation, subcellular localization and multimerization regulate its role in early embryogenesis.

Dishevelled (Dsh) induces a secondary axis and can translocate to the membrane when activated by Frizzleds; however, dominant-negative approaches have not supported a role for Dsh in primary axis formation. We demonstrate that the Dsh protein is post-translationally modified at the dorsal side of the embryo: timing and position of this regulation suggests a role of Dsh in dorsal-ventral patterning in Xenopus. To create functional links between these properties of Dsh we analyzed the influence of endogenous Frizzleds and the Dsh domain dependency for these characteristics. Xenopus Frizzleds phosphorylate and translocate Xdsh to the membrane irrespective of their differential ectopic axes inducing abilities, showing that translocation is insufficient for axis induction. Dsh deletion analysis revealed that axis inducing abilities did not segregate with Xdsh membrane association. The DIX region and a short stretch at the N-terminus of the DEP domain are necessary for axis induction while the DEP region is required for Dsh membrane association and its phosphorylation. In addition, Dsh forms homomeric complexes in embryos suggesting that multimerization is important for its proper function.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Interaction between Wnt and TGF-beta signalling pathways during formation of Spemann's organizer.

Members of the Wnt and TGF-beta superfamilies regulate both cell fate and proliferation during development and tissue maintenance. In the early amphibian embryo, the Wnt and TGF-beta superfamily signalling cascades are required for the establishment of a dorsal signalling centre, Spemann's organizer. Intracellular proteins of both pathways, upon activation, translocate to the nucleus to participate in transcription. Here we show that beta-catenin and Lef1/Tcf, which are downstream components of the Wnt signalling cascade, form a complex with Smad4, an essential mediator of signals initiated by members of the TGF-beta growth factor superfamily. In Xenopus, this interaction directly and synergistically affects expression of the twin (Xtwn) gene during formation of the organizer. This is, to our knowledge, the first demonstration of a physical interaction between TGF-beta and Wnt signalling components in vivo.

3T3 Cells↗

PubMed2000

Natriuretic peptide system in the rat lacrimal gland.

The presence and characteristics of the natriuretic peptides and their receptors in the rat exorbital lacrimal gland were investigated. Serial dilution curves of the gland extracts were parallel to the standard curves of synthetic atrial natriuretic peptide (ANP) or C-type natriuretic peptide (CNP). Immunoreactive ANP or CNP in the gland extracts co-eluted with authentic ANP or CNP, and their contents were 4.95 +/- 0.60 and 2.87 +/- 0.53 pg mg(-1)protein (quadruplicate), respectively. By immunohistochemistry, strong immunoreactivities of ANP and CNP were co-localized in the tubules and excretory ducts of the gland, and moderate immunoreactivities were found in the myoepithelial cells and acini. Productions of guanosine 3',5'-cyclic monophosphate by particulate guanylyl cyclase in the gland membranes were stimulated by natriuretic peptides in a dose-dependent manner, and that by CNP was larger than by ANP. Messenger RNAs for ANP, CNP and their receptors were detected by reverse transcription-polymerase chain reaction. These results indicate that natriuretic peptides and their specific receptors are found in the rat lacrimal gland. Therefore, it is suggested that natriuretic peptide system may play physiological roles in the rat lacrimal gland.

Animals↗

PubMed2000

Localization of receptors for natriuretic peptide and endothelin in the duct of the epididymis of the freshwater turtle.

The presence of receptor subtypes for natriuretic peptides (NPs) and endothelin (ET) in the epididymis of the freshwater turtle, Amyda japonica, was examined by quantitative in vitro autoradiography using iodinated mammalian-type atrial NP ((125)I-ANP((1-28))), phylogenically conserved C-type NP ((125)I-[Tyr(0)]-CNP((1-22))), and ET-1 ((125)I-ET-1) as radiolabeled ligands. To characterize NP receptor (NPR) subtypes, we also performed an activation of particulate guanylyl cyclase (GC) in membranes of the epididymis by NPs. Specific (125)I-ANP((1-28)) and (125)I-[Tyr(0)]-CNP((1-22)) bindings were localized in surrounding smooth muscle cell layer of the duct of the epididymis with an apparent dissociation constant (K(d)) of 0.84+/-0.15 and 1.74+/-0.39 nM and a maximal binding capacity (B(max)) of 0.47+/-0.11 and 0.08+/-0.01 fmol/mm(2), respectively. Bindings of (125)I-ANP((1-28)) and (125)I-[Tyr(0)]-CNP((1-22)) to these sites were also displaced by des[Gln(18),Ser(19),Gly(20), Leu(21),Gly(22)]ANF((4-23)), a specific ligand of the NP clearance receptor. Production of 3',5'-cyclic guanosine monophosphate by particulate GC in membranes of the epididymis was stimulated by ANP((1-28)), BNP((1-26)), and CNP((1-22)). Receptor subtypes for ET in the epididymis were characterized by competition with BQ 123 and BQ 788 as specific antagonists for ET receptors, type A (ET(A)) and type B (ET(B)) subtypes, respectively. Specific (125)I-ET-1 bindings were localized in the smooth muscle cell layer of the duct of the epididymis with K(d) and B(max) of 0.21+/-0.03 nM and 0.52+/-0.05 fmol/mm(2), respectively. These specific bindings were potently inhibited in a dose-dependent manner by BQ 123, whereas BQ 788 (10 microM) was not in competing for specific (125)I-ET-1 bindings in this structure. Therefore, these results indicate that specific NP and ET receptors are localized in surrounding smooth muscle cells of the duct of the epididymis of the freshwater turtle. It is also suggested that biological and clearance NPR-like subtypes coexist in these cells, and the predominant ET receptor subtype in this tissue is the ET(A)-like receptor. The localization of specific receptors for NPs and ET in the epididymis may be involved in the control of the transport of sperm in the freshwater turtle.

Animals↗

PubMed2000

Cyclosporine impairs the guanylyl cyclase activity of the natriuretic peptide receptor in the glomerulus.

In order to elucidate the involvement of the atrial natriuretic peptide (ANP) and its receptor (natriuretic peptide receptor; NPR) system in cyclosporine-induced nephrotoxicity, we investigated the cyclosporine A (CsA)-induced changes in characteristics of the NPR/guanylyl cyclase system in the glomerulus and inner medulla of the rat kidney. CsA was administered intramuscularly to rats for 2 weeks (CsA group). Particulate guanylyl cyclase activity was measured in glomerular and inner medullary membranes. For receptor characteristics, quantitative in vitro receptor autoradiography was performed. The guanylyl cyclase activity in the glomerulus from the CsA group was attenuated compared with that from the control. However, the activity in the inner medulla was not affected by CsA treatment. Direct application of CsA to normal glomerular membrane completely abolished the ANP-induced guanylyl cyclase activation. Binding studies, using(125)I-ANP, revealed that B(max)was decreased in the CsA group, while K(d)was not affected in the glomerulus. However, in the inner medulla, neither B(max)nor K(d)was affected by CsA treatment. CsA did not displace the(125)I-ANP bindings to NPRs in the normal rat kidney. Local tissue ANP as well as plasma ANP concentration in both groups was not significantly different. These results indicate that CsA impairs the guanylyl cyclase activity mainly in the glomerulus by the decrease in NPR population and/or by direct inhibition, suggesting that the ANP/NPR system might be involved in CsA-induced nephrotoxicity.

Animals↗

PubMed2000

Immunohistochemical localization of C-type natriuretic peptide in the rat submaxillary salivary gland.

To define the localization and characteristics of C-type natriuretic peptide (CNP) in the rat submaxillary gland, immunohistochemistry and gel permeation-high-performance liquid chromatography were used. Immunoreactive (IR)-CNP was localized in cells of the granular convoluted tubule, striated duct and endothelial cells of the capillary, where atrial natriuretic peptide (ANP) was colocalized in consecutive sections, but not in acini. Gland extracts co-eluted with synthetic CNP and its content was 60.3+/-4.9 pg/mg protein (n=4). Molecular profiles of immunoreactive material showed two peaks corresponding to synthetic CNP((1-53)) and CNP((1-22)). These results indicate that CNP is colocalized with ANP in the duct and endothelial cells of the rat submaxillary gland. Therefore, CNP may have a physiological role in the submaxillary gland by interacting with ANP and/or other biologically active substances in the ducts and granular convoluted tubule cells.

Animals↗

PubMed2000

New xenicane diterpenoids from the gorgonian Acalycigorgia inermis.

Acalycixeniolides D-G [corrected] (1-4), four new diterpenoids of the xenicane class, have been isolated from the gorgonian Acalycigorgia inermis. The structures of these compounds have been determined by combined spectroscopic methods. These compounds exhibited cytotoxicity against a human leukemia cell-line.

Animals↗

PubMed2000

New triterpenoid saponins from the sponge Melophlus isis.

Four new triterpenoid saponins were isolated, along with two known 30-norlanostane-type saponins- sarasinosides A(1) (1) and A(3) (2)-from the sponge Melophlus isis collected from Guam. The structures of these new compounds (3-6) proved to be 30-norlanosta-8(14),24-dien-23-ones bearing two hydroxyl groups or the corresponding methoxy groups at the 9alpha,15alpha- and 9alpha, 15beta-positions and the pentasaccharide portion identical with those of 1 and 2 at the 3beta position, by spectral, chemical, and GC analyses.

Animals↗

PubMed2000

Diminished adenylate cyclase activity and aquaporin 2 expression in acute renal failure rats.

BACKGROUND: The present study was aimed at investigating the changes of aquaporin 2 (AQP2) expression and its underlying mechanisms in ischemic acute renal failure (ARF). METHODS: ARF was induced by clamping the both renal arteries for 60 minutes in rats. Two or seven days later, AQP2 expression and trafficking were determined in the kidney by Western blot analysis and immunohistochemistry. The activity of adenylate cyclase was also measured. RESULTS: The urinary flow rates in ARF-2 and ARF-7 day were significantly increased in association with decreases of urine osmolality. While there was decreased expression of AQP2 in the cortex, outer medulla, and inner medulla in ARF, it was most pronounced in the outer medulla. The AQP2 expression was reduced in the apical membrane-enriched fraction as well the subapical vesicle-enriched fraction in ARF; however, the degree was greater in the former than in the latter. Immunohistochemical study also showed a markedly decreased expression of AQP2 in the collecting duct in ARF. cAMP generation in response to arginine vasopressin (AVP) in the kidney was attenuated in ARF, most prominently in the outer medulla. cAMP generation in the outer medulla in response to forskolin was not affected, but sodium fluoride was significantly blunted in ARF. CONCLUSIONS: The AVP-stimulated adenylate cyclase activity is impaired in ARF, secondary to a defect at the level of the G protein. The expression of AQP2 was reduced as a consequence, which may in part account for urinary concentration defect in ARF.

Acute Kidney Injury↗

PubMed2000

C-type natriuretic peptide inhibits ANP secretion and atrial dynamics in perfused atria: NPR-B-cGMP signaling.

The purpose of the present experiments was to define the role of C-type natriuretic peptide (CNP) in the regulation of atrial secretion of atrial natriuretic peptide (ANP) and atrial stroke volume. Experiments were performed in perfused beating and nonbeating quiescent atria, single atrial myocytes, and atrial membranes. CNP suppressed in a dose-related fashion the increase in atrial stroke volume and ANP secretion induced by atrial pacing. CNP caused a right shift in the positive relationships between changes in the secretion of ANP and atrial stroke volume or translocation of the extracellular fluid (ECF), which indicates the suppression of atrial myocytic release of ANP into the paracellular space. The effects of CNP on the secretion and contraction were mimicked by 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP). CNP increased cGMP production in the perfused atria, and the effects of CNP on the secretion of ANP and atrial dynamics were accentuated by pretreatment with an inhibitor of cGMP phosphodiesterase, zaprinast. An inhibitor of the biological natriuretic peptide receptor (NPR), HS-142-1, attenuated the effects of CNP. The suppression of ANP secretion by CNP and 8-BrcGMP was abolished by a depletion of extracellular Ca(2+) in nonbeating atria. Natriuretic peptides increased cGMP production in atrial membranes with a rank order of potency of CNP > BNP > ANP, and the effect was inhibited by HS-142-1. CNP and 8-BrcGMP increased intracellular Ca(2+) concentration transients in single atrial myocytes, and mRNAs for CNP and NPR-B were expressed in the rabbit atrium. From these results we conclude that atrial ANP release and stroke volume are controlled by CNP via NPR-B-cGMP mediated signaling, which may in turn act via regulation of intracellular Ca(2+).

Animals↗

PubMed2000

Distinct roles for L- and T-type Ca(2+) channels in regulation of atrial ANP release.

Atrial secretion of atrial natriuretic peptide (ANP) has been shown to be regulated by atrial workload. Although modulating factors for the secretion of ANP have been reported, the role for intracellular Ca(2+) on the secretion of ANP has been controversial. The purpose of the present study was to define roles for L- and T-type Ca(2+) channels in the regulation of ANP secretion in perfused beating rabbit atria. BAY K 8644 (BAY K) increased atrial stroke volume and pulse pressure. BAY K suppressed ANP secretion and ANP concentration in terms of extracellular fluid (ECF) translocation concomitantly with an increase in atrial dynamics. BAY K shifted the relationship between ANP secretion and ECF translocation downward and rightward. These results indicate that BAY K inhibits myocytic release of ANP. In the continuous presence of BAY K, diltiazem reversed the effects of BAY K. Diltiazem alone increased ANP secretion and ANP concentration along with a decrease in atrial dynamics. Diltiazem shifted relationships between ANP secretion and atrial stroke volume or ECF translocation leftward. The T-type Ca(2+) channel inhibitor mibefradil decreased atrial dynamics. Mibefradil inhibited ANP secretion and ANP concentration in contrast with the L-type Ca(2+) channel inhibitor. These results suggest that activation of L- and T-type Ca(2+) channels elicits opposite effects on atrial myocytic release of ANP.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed2000

Renin angiotensin system of rabbit clitoral cavernosum: interaction with nitric oxide.

PURPOSE: Angiotensin (ANG) II has been known to be a potent modulator for the maintenance of smooth muscle tone of the penile cavernosum. However, its role in clitoral cavernosum is unknown. The clitoris is the homologue of the penis arising from the embryological genital tubercle. We investigated the presence of ANG II receptors, the function of ANG II, and its interaction with nitric oxide (NO) in rabbit clitoral cavernosum. MATERIALS AND METHODS: The isometric tension was measured in the strips of clitoral cavernosum. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to evaluate expression of AT1a and AT1b ANG II receptor subtype mRNAs. In vitro autoradiography was used to localize ANG II receptors in the clitoral cavernosum. RESULTS: The clitoral cavernosum was contracted dose-dependently by the addition of ANG II. Dup 753 (ANG II type 1 receptor antagonist) inhibited significantly ANG II induced contraction. PD 123,319 (ANG II type 2 receptor antagonist) did not affect the ANG II response. Pretreatment with NG-nitro-L-arginine methyl ester (NO synthase inhibitor) accentuated contractions induced by ANG II. Specific binding sites for 125I-ANG II were found in the clitoral cavernosum. The dissociation constant (Kd) was 0.58 + or - 0.05 nM. Specific binding of 125I-ANG II was displaced by Dup 753 (10-5 M) but not by PD 123,319 (10-5 M). The inhibitory constant (Ki) for Dup 753 was 23. 4 + or - 9.73 nM and mRNAs for AT1a and AT1b receptor subtypes were detected by RT-PCR. CONCLUSION: The present study shows that ANG II is involved in the regulation of clitoral cavernosum smooth muscle tone via ANG II receptor subtype AT1, and that ANG II has cross-talk with NO.

Angiotensin I↗

PubMed2000

Coexistence of C-type natriuretic peptide and atrial natriuretic peptide systems in the bovine cornea.

PURPOSE: To determine whether the cornea synthesizes natriuretic peptides and contains their receptors. METHODS: The synthesis of the natriuretic peptides, C-type natriuretic peptide (CNP) and atrial natriuretic peptide (ANP), in the bovine cornea was determined by high-performance liquid chromatography (HPLC) with radioimmunoassay and Southern blot analysis. The presence of natriuretic peptide receptor (NPR)-A and -B and their localizations were measured by reverse transcription-polymerase chain reaction (RT-PCR), in vitro autoradiography, and the activation of particulate guanylyl cyclase by natriuretic peptides in the corneal membrane. RESULTS: The serial dilution curves of corneal extracts were parallel to the standard curves of CNP and ANP. With reversed-phase HPLC, a major immunoreactive peak of CNP or ANP was observed at the elution time corresponding with synthetic CNP(1-53) or atriopeptin III (APIII), respectively. The presence of mRNAs of CNP and ANP was also detected in the cornea by RT-PCR and/or Southern blot analysis. Production of 3',5'-cyclic guanosine monophosphate (cGMP) by the activation of particulate guanylyl cyclase in the corneal membrane was stimulated by ANP, BNP, and CNP. More cGMP was produced by CNP than by the other natriuretic peptides. Specific 125I-[Tyr0]-CNP(1-22) binding sites were localized in the endothelial cell layer of cornea. The apparent dissociation constant (Kd) value of the cornea was 3.06 +/- 0.73 nM and the maximum binding capacity was 3.40 +/- 0.63 femtomoles/mm2. Both NPR-A and NPR-B mRNAs were detected by RT-PCR. CONCLUSIONS: The cornea synthesizes CNP and ANP and contains their receptors. These results suggest that the CNP and ANP systems coexist in the bovine cornea.

Animals↗

PubMed1999

Mammalian BMP-1/Tolloid-related metalloproteinases, including novel family member mammalian Tolloid-like 2, have differential enzymatic activities and distributions of expression relevant to patterning and skeletogenesis.

Vertebrate bone morphogenetic protein 1 (BMP-1) and Drosophila Tolloid (TLD) are prototypes of a family of metalloproteases with important roles in various developmental events. BMP-1 affects morphogenesis, at least partly, via biosynthetic processing of fibrillar collagens, while TLD affects dorsal-ventral patterning by releasing TGFbeta-like ligands from latent complexes with the secreted protein Short Gastrulation (SOG). Here, in a screen for additional mammalian members of this family of developmental proteases, we identify novel family member mammalian Tolloid-like 2 (mTLL-2) and compare enzymatic activities and expression domains of all four known mammalian BMP-1/TLD-like proteases [BMP-1, mammalian Tolloid (mTLD), mammalian Tolloid-like 1 (mTLL-1), and mTLL-2]. Despite high sequence similarities, distinct differences are shown in ability to process fibrillar collagen precursors and to cleave Chordin, the vertebrate orthologue of SOG. As previously demonstrated for BMP-1 and mTLD, mTLL-1 is shown to specifically process procollagen C-propeptides at the physiologically relevant site, while mTLL-2 is shown to lack this activity. BMP-1 and mTLL-1 are shown to cleave Chordin, at sites similar to procollagen C-propeptide cleavage sites, and to counteract dorsalizing effects of Chordin upon overexpression in Xenopus embryos. Proteases mTLD and mTLL-2 do not cleave Chordin. Differences in enzymatic activities and expression domains of the four proteases suggest BMP-1 as the major Chordin antagonist in early mammalian embryogenesis and in pre- and postnatal skeletogenesis.

Amino Acid Sequence↗

PubMed1999

Bone morphogenetic protein antagonism of Spemann's organizer is independent of Wnt signaling.

The Xenopus homeobox gene twin is involved in the Wnt-mediated induction of Spemann's organizer. Additionally, several lines of evidence indicate that bone morphogenetic proteins (BMPs) play a role in repressing the formation of the organizer by antagonizing the expression of genes involved in organizer establishment. In order to determine at what level BMPs exert their effect, we measured the activity of different genes expressed within the organizer region. We report that BMP signaling can antagonize the induction of the dorsal-specific gene goosecoid but is unable to affect Wnt signaling at the level of twin. These results suggest that the antagonistic activities of BMPs in organizer formation occur postzygotically, independent of twin regulation, and that Wnt-like dorsal determinant signaling pathways do not crosstalk with BMPs.

Animals↗

PubMed1999

Activation of particulate guanylyl cyclase by Vibrio vulnificus hemolysin.

Recently we reported that Vibrio vulnificus hemolysin, an exotoxin produced by V. vulnificus, dilates rat thoracic aorta via elevated cGMP levels without affecting nitric oxide synthase. We investigated the mechanism further by observing the guanylyl cyclase activities in cytosolic, membrane, unfractionated, or reconstituted preparations. Hemolysin did not activate guanylyl cyclase in the membrane or cytosolic fraction, while it activated guanylyl cyclase in unfractionated or reconstituted preparation. The increased activity was not inhibited by the HS-142-1, a microbial polysaccharide which antagonizes atrial natriuretic peptide receptor, or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a soluble guanylyl cyclase inhibitor. However, it was attenuated by 6-(phenylamino)-5,8-quinolinedione (LY 83.583), which inhibits the catalytic domain of both guanylyl cyclases, and by cholesterol, which blocks hemolysin-incorporation into the membrane. Removing ATP, a cofactor of particulate guanylyl cyclase, attenuated the activation and ATPgammaS, a non-phosphorylating analog, restored it. These results suggest that V. vulnificus hemolysin activates particulate guanylyl cyclase via hemolysin incorporation into the vascular smooth muscle cell membrane in cooperation with certain unidentified cytosolic component(s).

Adenosine Triphosphate↗