Disruption of human limb morphogenesis by a dominant negative mutation in CDMP1.

Chondrodysplasia Grebe type (CGT) is an autosomal recessive disorder characterized by severe limb shortening and dysmorphogenesis. We have identified a causative point mutation in the gene encoding the bone morphogenetic protein (BMP)-like molecule, cartilage-derived morphogenetic protein-1 (CDMP-1). The mutation substitutes a tyrosine for the first of seven highly conserved cysteine residues in the mature active domain of the protein. We demonstrate that the mutation results in a protein that is not secreted and is inactive in vitro. It produces a dominant negative effect by preventing the secretion of other, related BMP family members. We present evidence that this may occur through the formation of heterodimers. The mutation and its proposed mechanism of action provide the first human genetic indication that composite expression patterns of different BMPs dictate limb and digit morphogenesis.

Regulation of the mGluR5, EAAT1 and GS expression by glucocorticoids in MG-63 osteoblast-like osteosarcoma cells.

INTRODUCTION: Growth factors, cytokines, sex steroid hormones and glucocorticoids have differential and complex effects on skeletal metabolism. Recently, the presence of the glutamatergic (Glu) system in bone cells has provided new evidence for its possible role in bone physiology. Consequently, we have investigated the regulation of certain components of the Glu system by glucocorticoids in MG-63 osteoblast-like osteosarcoma cells, in vitro. MATERIALS AND METHODS: We characterized the effects of dexamethasone on the expression of the mGluR5, EAAT1 and GS, at mRNA and protein level, using relative quantitative RTPCR and Western blot analysis, respectively. RESULTS: We confirmed the induction of GS expression by dexamethasone published previously. In addition, we documented for the first time the expression of the mGluR5 and EAAT1 in MG-63 cells, as well as the ability of dexamethasone to upregulate the expression of the mGluR5 and EAAT1 in the MG-63 cells. CONCLUSIONS: Components of the glutamatergic system may play a role in bone pathophysiology.

Carotid body paraganglioma and SDHD mutation in a Greek family.

BACKGROUND: Carotid body (CB) is a highly specialized paraganglion originating from the neural crest ectoderm. CB paraganglion can be caused either by a genetic predisposition (hereditary paraganglia) or by chronic hypoxic stimulation. Germline mutations in any of the following genes: SDHD, SDHC, SDHB, PGL2 or other unknown genes, can cause paragangliomas (PGLs). MATERIALS AND METHODS: We studied a Greek family in which the two daughters had carotid body paraganglioma, whereas both parents did not. RNA extraction, reverse transcriptase polymerase chain reaction and direct DNA sequencing were performed, in order to identify SDHD mutations in all four exons. RESULTS: Our results revealed the existence of the missense mutation Y114C, in exon-4 of the SDHD gene, in the unaffected father and both affected sisters. CONCLUSION: DNA testing was performed, for the first time in Greece, on patients with carotid body tumor. This marks a new geographical location, in the literature, for this mutation.

Repetitive and site-specific molecular staging of prostate cancer using nested reverse transcriptase polymerase chain reaction for prostate specific antigen and prostate specific membrane antigen.

We performed repetitive molecular staging using, nested rt-PCR for PSA and PSM at the peripheral blood (PB) and bone marrow (BM) of patients with prostate cancer (Pr.Ca) and benign prostate hyperplasia (BPH) after transrectal ultrasonography-guided biopsy (TRUS-B; 6-9 biopsies/patient), Pr.Ca patients after radical prostatectomy (RP), and Pr.Ca patients with diffuse bony metastases. All BPH patients (N = 20) tested negative at BM. Of the 2 who tested positive at PB 2 weeks after TRUS-B tested negative 8 weeks after TRUS-B. Of the 17 Pr.Ca, 7 (41.2%) tested positive at PB for PSA and PSM 2 weeks after TRUS-B while only 4 (23.5%) of them tested positive at repetitive analysis 8 weeks after TRUS-B. Two (11.8%) of the 17 Pr.Ca patients had positive analysis at BM for PSA and PSM 2 and 8 weeks after TRUS-B. Of 12 Pr.Ca patients with negative pre-operative molecular staging, 7 (58.3%) tested positive at PB for PSA and PSM 2 months post-RP but only 3 (25%) of them re-tested positive 12 months post-RP. Of these 12 Pr.Ca, 4 (33.3%) tested positive at BM for PSA and PSM 2 months post-RP while none re-tested positive 12 months post-RP. All Pr.Ca (N = 20) with diffuse bony lesions tested positive at BM. At PB, 6 of them (30%) tested negative for both PSA and PSM. Our data suggest that nested rt-PCR for PSA and PSM at PB is affected by TRUS-B and RP, while such analysis at BM concerted diffuse bony disease.

Novel approach to the molecular diagnosis of Marfan syndrome: application to sporadic cases and in prenatal diagnosis.

Marfan syndrome is an autosomal dominant disorder affecting the skeletal, ocular, and cardiovascular systems. Defects in the gene that encodes fibrillin-1 (FBN1), the main structural component of the elastin-associated microfibrils, are responsible for the disorder. Molecular diagnosis in families with Marfan syndrome can be undertaken by using intragenic FBN1 gene markers to identify and track the disease allele. However, in sporadic cases, which constitute up to 30% of the total, DNA-based diagnosis cannot be performed using linked markers but rather requires the identification of the specific FBN1 gene mutation. Due to the size and complexity of the FBN1 gene, identification of a causative Marfan syndrome mutation is not a trivial undertaking. Herein, we describe a comprehensive approach to the molecular diagnosis of Marfan syndrome that relies on the direct analysis of the FBN1 gene at the cDNA level and detects both coding sequence mutations and those leading to exon-skipping, which are often missed by analysis at the genomic DNA level. The ability to consistently determine the specific FBN1 gene mutation responsible for a particular case of Marfan syndrome allows both prenatal and pre-implantation diagnosis, even in sporadic instances of the disease.

Conversion of nested reverse-transcriptase polymerase chain reaction from positive to negative status at peripheral blood during androgen ablation therapy is associated with long progression-free survival in stage D2 prostate cancer patients.

BACKGROUND: Nested reverse-transcriptase polymerase chain reaction (nested rt-PCR) for the detection of mRNA of prostate specific antigen (PSA) and prostate specific membrane antigen (PSMA) in peripheral blood samples (PB) and bone marrow biopsies (BM) can assess the extraprostatic growth of prostate cells. MATERIALS AND METHODS: Nested rt-PCR for PSA and PSMA at PB and BM was performed (a) at diagnosis, (b) after 6 months from the initiation of combined androgen blockade [(CAB); triptorelin 3.75 mg, i.m., q28 days plus flutamide 250 mg, per os, tid] and, (c) at progression to androgen refractory stage in 28 patients with newly-diagnosed stage D2 prostate cancer. RESULTS: At diagnosis, all patients were found to be rt-PCR positive for PSA and PSMA at BM while 7 (25%) were rt-PCR negative for PSA and PSMA at PB. Nine out of 21 patients with rt-PCR-positive status have converted to rt-PCR-negative status at PB during CAB while they remained rt-PCR-positive at BM. The rt-PCR-negative status at PB during CAB was associated with progression-free survival >12 months (p=0.029). At progression to androgen refractory stage all but 2 patients were rt-PCR-positive at PB for PSA and PSMA, among them 3 who were rt-PCR-negative at diagnosis. CONCLUSION: Our data suggest that conversion to rt-PCR-negative status at PB for PSA and PSMA during objective clinical response to CAB is associated with long progression-free survival in stage D2 disease.

Cancer and bone repair mechanism: clinical applications for hormone refractory prostate cancer.

It is a long-standing clinical observation that the bone corresponds to the prevalent site for metastatic growth of prostate cancer. In addition, bone metastases of this malignancy produce a potent blastic reaction, in contrast to the overwhelming majority of other osteotropic neoplasms, whose metastases are generally associated with an osteolytic reaction. Osteoblastic metastases represent almost always the first and, frequently, the exclusive site of disease progression to hormone refractory stage, stage D3. Moreover, the number of skeletal metastatic foci is the most powerful independent prognostic factor associated with a limited response to hormone ablation therapy and poor survival of advanced prostate cancer. It is noteworthy that disease progression to hormone refractory stage occurs almost always in osteoblastic metastases. These clinical observations suggested that the osteoblastic reaction is possibly not an innocent bystander of the metastatic prostate tumour growth, simply suffering its consequences, but it may in fact facilitate the efforts of metastatic cells to expand their population. An extensive line of research in the pathophysiology of osteoblastic metastases has established that the local blastic reaction involves the uPA/plasmin/IGF/IGFBP-3/TGFbs bioregulation system which can stimulate both the growth of osteoblasts and prostate cancer cells. Furthermore, we were the first to characterize osteoblast-derived 'survival factors' able to rescue metastatic prostate cancer cells from chemotherapy-induced apoptosis. These data resulted in the development of a novel concept of an anti-survival factor therapy, namely an anti-IGF-1 therapy, which has provided encouraging preliminary data in a phase II clinical trial with terminally-ill hormone/chemotherapy-resistant prostate cancer patients.

Three-dimensional type I collagen co-culture systems for the study of cell-cell interactions and treatment response in bone metastases.

The available monolayer culture systems for the study of bone metastases constitute a suboptimal simulation of the in vivo pathophysiology of bone metastases, and therefore, do not provide sufficient information to assess the morphologic evidence of bone reaction to cancer cells, the nature of cell-specific mediators of osteolysis and osteoplasia and the response to treatment. Therefore, we have developed a three-dimensional (3-D) type I collagen gel system that allows co-culture of human osteoblasts (MG-63) with cancer cells, such as MCF-7, MDA-MB-231 or ZR-75 breast cancer cells, PC-3 prostate cancer, KLE endometrial cancer cells and Calu-1 lung cancer cells. We used type I collagen purified from rat tail tendons and the 3-D system was prepared by mixing MG-63 cells with type I collagen in 24-well plates. The 3-D system was inoculated with cancer cells and processed with standard cell culture procedures. After 1 week of culture, the matrix gel was fixed with formalin and embedded in paraffin. Serial sections were stained with trichrome Masson stain and modified Masson-Goldner stain, as well as analyzed by in situ hybridization, immunohistochemistry and the TUNEL technique for semi-quantitative detection of apoptotic cell death, assessing the response to adriamycin therapy. The inoculation of PC-3 cells in this collagen matrix produced a blastic reaction, documented by an increased number of MG-63 cells and increased density of type I collagen. The human KLE cells and inoculation of cell-free media produced no reaction, while ZR-75, MCF-7 and Calu-1 cells produced local degradation of the collagen matrix. In situ hybridization revealed the expression of Insulin-like growth factor 1 (IGF-1) and urokinase-type plasminogen activator (uPA) mRNA, while immunohistochemistry detected differential expression of uPA and cathepsin D. Adriamycin induced apoptotic cell death in prostate cancer cells and estrogen receptor negative (ER-) MDA-MB-231 breast cancer cells, while adriamycin did not induce apoptosis but cytostasis in ER+ MCF-7 cells. The adriamycin-induced apoptosis was inhibited by co-culture with osteoblast-like cells (MG-63). We conclude that this 3-D culture system is a useful in vitro model allowing the analysis of local mediators of osteolytic and osteoblastic reactions to bone metastases and treatment response.

Skeletal growth factor involvement in the regulation of fracture healing process.

Skeletal growth factors are peptides that serve as signalling agents for living cells, thereby participating in the autocrine, paracrine, intracrine and endocrine bioregulation of tissues and organs in human physiology. Growth factors elicit their cellular actions after binding to specific receptors, which are large transmembrane proteins located on target cells. These receptors relay signals via specific intracellular signal transduction pathways capable of regulating gene transcription, thereby modifying cell proliferation, cell function, cell differentiation and apoptosis. Notably, growth factors and their specific receptors are expressed in and around a bone fracture repair site, suggesting strongly that they play a significant role in the physio/pathology of fracture healing. Conceivably, fine adjustments of specific growth factor activity during the different stages of the fracture healing process can serve as potential therapeutic targets, enhancing bone repair capacity and reducing irregularities of the healing process.

Three-dimensional type I collagen cell culture systems for the study of bone pathophysiology.

Three-dimensional (3-D) type I collagen cell culture systems composed of reconstituted collagen fibres are able to support short- and long-term growth of various cell types, including cancer cell lines, endothelial cells, endometrial cells, hepatocytes, osteoblasts and fibroblasts and to sustain or even enhance cell differentiation, in vitro. In addition, 3-D culture systems have been successfully used in the investigation of complex biological processes, such as angiogenesis, wound healing, tumour invasion and metastasis. The latter suggested that 3-D culture systems have the potential to simulate cell-cell interactions, which take place in tissues under physiological and pathophysiological conditions. This review focuses on the investigational use of 3-D collagen cell culture systems in bone physiology and the pathophysiology of skeletal metastasis.

CD40 expression and its association with low-grade inflammation in a Greek population of type 1 diabetic juveniles: evidence for differences in CD40 mRNA isoforms expressed by peripheral blood mononuclear cells.

BACKGROUND: CD40 signalling has been associated with the pathogenesis of autoimmune diseases and diseases with low-grade chronic inflammation. OBJECTIVE: To investigate, early in the course of type 1 diabetes (T1DM) patients, the expression of CD40 system components, as well as to explore the association of plasma and urine concentrations of CD40 with known inflammatory markers in T1DM. METHODS: Plasma, urine and peripheral blood mononuclear cells (PBMCs) from 70 T1DM patients without clinically detected chronic complications and 40 healthy controls (HCs) were examined using ELISA, western-blot, semi-quantitative RT-PCR and DNA-sequencing. RESULTS: Patients had significantly higher plasma soluble CD40 (sCD40) levels associated with higher Interleukin-6 (IL-6), matrix metalloproteinase-9 (MMP-9) and CRP levels compared with healthy controls. This difference was also evident between poorly and well-controlled diabetic patients. The elevated plasma sCD40 levels do not appear to be due to diminished renal excretion since sCD40 concentrations in the urine were also elevated, suggesting an increased CD40 production. An upregulation of PBMCs' CD40 was evident in T1DM patients associated with higher sCD40, IL-6 and CRP levels. Furthermore, the main CD40 isoform (isoform-I) was solely expressed in poorly controlled diabetics' PBMCs, who also demonstrated cellular CD40 upregulation, higher plasma CD40, CRP, IL-6 and MMP-9 levels compared with the well-controlled diabetics and the control group, who co-expressed type I and II isoforms. CONCLUSIONS: Homeostatic dysregulation of CD40 and its association with inflammatory markers in T1DM patients, especially in those with poor glycaemic control, implies a pathophysiological role of CD40 in the low-grade inflammatory process in T1DM.

Molecular diagnosis of the viral component in cardiomyopathies: pathophysiological, clinical and therapeutic implications.

BACKGROUND: Myocarditis is defined as the inflammation of myocardium associated with cardiac dysfunction. Despite this clear-cut definition, diagnosis and etiologic treatment continue to create considerable debate. Viral infections are frequent causes of myocarditis and there is evidence that persistent viral infection is associated with poor prognosis in different subtypes of cardiomyopathy. OBJECTIVE: To review methods for diagnosis of viral myocarditis and present the use of polymerase chain reaction (PCR)-based protocols for evaluating viral infection in myocarditis/cardiomyopathies. METHODS: A review of published literature. RESULTS/CONCLUSION: There is increasing evidence that PCR-based protocols can provide reliable molecular evidence for the presence of viral infection in myocardium. Thus application of molecular techniques will allow collection and analysis of more information on the epidemiology of viral cardiomyopathies, patient risk stratification and appropriate medical treatment.

Bone microenvironment-related growth factors, zoledronic acid and dexamethasone differentially modulate PTHrP expression in PC-3 prostate cancer cells.

Bone metastasis microenvironment-related growth factors such as insulin-like growth factor 1 (IGF-1), transforming growth factor beta 1 (TGF-beta1), basic fibroblast growth factor (bFGF) and interleukin 6 (IL-6) show survival factor activity, thereby inhibiting chemotherapy-induced apoptosis of PC-3 prostate cancer cells in vitro. Recently, zoledronic acid has been shown to induce apoptosis in PC-3 prostate cancer cells while overexpression of parathyroid hormone-related protein (PTHrP) inhibits serum deprivation-induced apoptosis in PC-3 cells. Consequently, we have investigated whether IGF-1, TGF-beta1, bFGF, IL-6, zoledronic acid and/or dexamethasone affect the expression of the PTHrP and type I PTH/PTHrP receptor (PTH.1R) in PC-3 prostate cancer cells using relative quantitative PCR and real-time PCR (expression at mRNA level) and immunocytochemical and immunofluorescence analysis (expression at protein level). Our data show that IGF-1, TGF-beta1, bFGF and IL-6 increase PTHrP mRNA expression and its perinuclear localization, while zoledronic acid (50 muM, 100 muM for 24 h and 48 h) and dexamethasone suppress PTHrP expression in PC-3 cells. We did not detect any appreciable change of the PTH.1R expression due to IGF-1, TGF- beta1, bFGF, IL-6, zoledronic acid or dexamethasone in PC-3 cells. Therefore, it is conceivable that bone metastasis microenvironment-related survival factor/anti-apoptotic activity and zoledronic acid anticancer action/pro-apoptotic activity on PC-3 cells is mediated, at least in part, by differential modulation of PTHrP expression.

Effect of protein kinase C inhibitors on Cl- conductance required for volume regulation after L-alanine cotransport.

We used electronic cell sizing and Cl- efflux measurements in guinea pig jejunal enterocytes to study activation of Cl- conductance under two experimental conditions, regulatory volume decrease (RVD) after passive hypotonic swelling and volume regulation during Na(+)-alanine cotransport. RVD after a hypotonic (0.5 x isotonic) challenge was not affected by the protein kinase C (PKC) inhibitor 100 microM 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7). Volume decrease after cell swelling in response to L-Ala (25 mM) was prevented by H-7 (P less than 0.05) or the more potent PKC inhibitor 10 nM staurosporine (P less than 0.001). L-Ala stimulated biphasic 36Cl efflux, a rapid efflux over 60 s which was inhibited by H-7 (P less than 0.01) and the Cl(-)-channel blocker anthracene-9-carboxylic acid (9-AC) (P less than 0.005). In contrast, after hypotonic dilution the rate of 36Cl efflux increased (P less than 0.005); H-7 had no effect but 9-AC inhibited the increase (P less than 0.01). Gramicidin (0.5 microM) added to cells maximally swollen by L-Ala in Cl(-)-containing medium caused 2 degree swelling (P less than 0.001), but 10 nM staurosporine reduced this 2 degree swelling (P less than 0.001). Addition of phorbol ester or synthetic diacylglycerol to villus cells under isotonic conditions, after gramicidin addition, caused cell swelling (P less than 0.005) that was inhibited by staurosporine (P less than 0.05). We concluded that PKC does not activate Cl- conductance for hypotonic RVD but that Na(+)-nutrient cotransport is a physiological stimulus for PKC to activate Cl- conductance necessary for volume regulation.

Isotonic volume reduction associated with cAMP stimulation of 36Cl efflux from jejunal crypt epithelial cells.

To determine the effect of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) on the isotonic volume of jejunal crypt epithelial cells, we isolated these cells by sequential perfusion using a hyperosmolar Ca(2+)-free solution and measured cell volume electronically. 8-BrcAMP caused rapid shrinkage to a reduced but stable cell volume; this isotonic volume reduction was prevented by either a Cl(-)-channel blocker, anthracene-9-carboxylate (A-9C), or Ba2+, a K(+)-channel blocker. 8-BrcAMP substantially increased the rate of A-9C-sensitive 36Cl efflux from crypt cells; this increased rate of efflux was not influenced by Ba2+ but was abolished by alterations in membrane potential. Following 8-BrcAMP-stimulated isotonic volume reduction, addition of either Ba2+ or A-9C caused the crypt cells to reswell. In contrast, 8-BrcAMP added to enterocytes isolated from the villus compartment did not result in A-9C-sensitive volume reduction or in an increased rate of 36Cl efflux. Our data demonstrate that epithelial cells isolated from jejunal crypt compartments respond directly to cAMP with a rapid volume reduction that is paralleled by an increase in 36Cl efflux through a conductive pathway. Unlike other Cl- secretory epithelial cells, the intestinal crypt cell does not appear to regulate its volume in an isotonic medium after cAMP-induced shrinkage.

Differences in Ca(2+)-mediation of hypotonic and Na(+)-nutrient regulatory volume decrease in suspensions of jejunal enterocytes.

We determined differences in the Ca2+ signalling of K+ and Cl- conductances required for Regulatory Volume Decrease (RVD) in jejunal villus enterocytes passively swollen (0.5 or 0.95.isotonic) compared with swelling because of the absorption of D-glucose (D-Glc) or L-Alanine (L-Ala). Cell volume was measured using electronic cell sizing. In nominally Ca(2+)-free medium containing EGTA (100 microM) RVD after 0.5 or 0.95.isotonic challenge was prevented. L-Ala swelling and subsequent RVD was influenced in Ca(2+)-free medium. Villus cells were incubated with 10 microM of the acetomethoxy derivative of 1,2.bis (2-aminophenoxy) ethane N,N,N1,N1 tetracetic acid (BAPTA-AM) and RVD after 0.5.isotonic swelling or L-Ala swelling was prevented. Niguldipine (0.1 microM), nifedipine (5 microM), diltiazem (100 microM), Ni2+, and Co2+ (1 mM) all prevented hypotonic RVD but had no effect on RVD after L-Ala addition. Charybdotoxin (25 nM) a potent inhibitor of Ca(2+)-activated K+ channels, had no effect on hypotonic RVD but prevented RVD of villus cells swollen by D-Glc. We used the calmodulin antagonists, naphthalene sulfonamide derivatives W-7 and W-13, to assess calmodulin activation of K+ and Cl- conductance in these two models. L-Ala swelling and subsequent RVD was not influenced by 25 microM W-7; hypotonic RVD was prevented by 25 microM W-7 or 100 microM W-13. The W-13 inhibition of RVD was by-passed with 0.5 microM gramicidin. Our data show that hypotonic RVD requires extracellular Ca2+ and that the K+ conductance activated is not charybdotoxin sensitive but requires calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of osmotic swelling on K+ conductance in jejunal crypt epithelial cells.

To further elucidate differences in ion transport properties between jejunal crypt and villus cells, we compared the responses of purified cell suspensions to hypotonic stress using electronic cell sizing to evaluate volume changes and 86Rb and 36Cl efflux. After hypotonic swelling, villus enterocytes undergo a regulatory volume decrease (RVD) due to the loss of K+ and Cl- through volume-activated conductances. After 0.6x isotonic challenge in Na(+)-free medium, crypt cells exhibited only partial RVD, with t1/2 congruent to 15 min. The addition of a cation ionophore, gramicidin (0.25 microM), to hypotonically swollen crypt cells caused an accelerated RVD, which was complete with t1/2 congruent to 5 min. Crypt epithelial cells showed no volume-activated 86Rb efflux, but villus enterocytes had an increased rate of 86Rb efflux after hypotonic dilution (P less than 0.001). Gramicidin added to hypotonically diluted crypt cells greatly increased the rate of 86Rb efflux compared with controls. Both villus (30 s; P less than 0.005) and crypt (2 min; P less than 0.001) cells exhibited volume-activated 36Cl efflux in absence of gramicidin. Cl- channel blockers anthracene-9-carboxylate (9-AC, 300 microM) and indanyloxyacetic acid (IAA-94, 100 microM) prevented crypt RVD (P less than 0.001) in the presence of gramicidin. Ouabain (P less than 0.001) or K(+)-free Na(+)-containing medium, but not Ba2+ (5 mM) or quinine (100 microM), prevented crypt partial RVD. We conclude that crypt cells lack volume-activated K+ conductance. The RVD exhibited by crypt cells, although partial, was due to Cl- loss through a volume-activated Cl- conductance and Na+ loss via Na(+)-K(+)-ATPase.

Isolation of a member of the neurotoxin/cytotoxin peptide family from Xenopus laevis skin which activates dihydropyridine-sensitive Ca2+ channels in mammalian epithelial cells.

We have used a sensitive bioassay of calcium-mediated volume changes in mammalian absorptive intestinal epithelial cells to screen extracts of the skin of the amphibian Xenopus laevis for the presence of factors affecting ion transport. A 66-residue peptide, purified using reversed-phase high performance liquid chromatography techniques, caused isotonic volume reduction of guinea pig jejunal villus cells in suspension. This volume reduction required extracellular Ca2+ and was prevented by the dihydropyridine-sensitive Ca2+ channel blocker niguldipine. Structural analysis demonstrated the presence of eight cysteines and a primary structure homologous to that of the neurotoxin/cytotoxin family found in the venom of certain poisonous snakes. The structure of the peptide was identical to that of xenoxin-1 purified from dorsal gland secretions of X. laevis (Kolbe, M., Huber A., Cordier, P., Rasmussen, U., Bouchon, B., Jaquinod, M., Blasak, R., Detot, E., and Kreil, G. (1993) J. Biol. Chem. 268, 16458-16464). Xenoxin-1 (10 nM) caused volume changes that required extracellular Ca2+ and were comparable in magnitude and direction to changes caused by BayK-8644 (100 nM), a dihydropyridine-sensitive Ca2+ channel agonist. The initial rate of dihydropyridine-sensitive 45Ca2+ influx was substantially increased by xenoxin-1. Staurosporine (10 nM) prevented volume changes caused by ATP (250 microM) but had no effect on volume changes caused by BayK-8644 or xenoxin-1. We conclude that xenoxin-1 directly activated dihydropyridine-sensitive Ca2+ channels in villus cells and that a mammalian homologue to xenoxin-1 may exist.

Sodium-bicarbonate cotransport in guinea pig ileal crypt cells.

Prior studies show that ileal HCO3- secretion is of crypt origin, possibly involving Na+-HCO3- cotransport. To test for the latter, we isolated crypt cells from guinea pig ileum and determined effects of medium HCO3-, Na+, K+, disulfonic stilbenes, and gramicidin on intracellular pH [pHi;2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein fluorescence], cell volume (electronic sizing), and Na+ efflux from 22Na+ -preloaded cells. Ileal crypt cells alkalinized when placed in sodium gluconate-HCO3- medium containing N-5-methyl-5-isobutyl amiloride (1 microM), bumetanide (10 microM) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (250 microM which blocks Cl-/HCO3- exchange but not Na+ dependent HCO3- uptake). Depolarization with either gramicidin (50 microM) or 50 mM K+ caused a further 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS)-inhibitable increase in pHi. Gramicidin also caused SITS-inhibitable cell swelling. Both gramicidin effects were Na+ dependent: at 0 mM Na+, gramicidin acidified and did not alter cell volume; at 25 mM, gramicidin also acidified; at 90 and 140 mM, gramicidin alkalinized and induced cell swelling. HCO3- -dependent SITS-inhibitable Na+ efflux from 22Na+ -preloaded cells was also seen. We conclude that ileal crypt cells engage in electrogenic Na+ -HCO3- symport.

The isolation and characterization of a novel corticostatin/defensin-like peptide from the kidney.

We report the isolation and characterization of RK-1, a novel peptide found in the kidney. RK-1 is related to the corticostatin/defensins and has the sequence MPC-SCKKYCDPWEVIDGSCGLFNSKYCCREK but differs from the very cationic corticostatins/defensins in having only one arginine and a calculated charge at pH 7 of +1. Like some myeloid corticostatin/defensins RK-1 inhibits the growth of Escherichia coli. Since corticostatin/defensins effect ion flux in responsive epithelia we used volume changes in villus enterocytes as a model system to study the effects of RK-1 on ion channels in epithelial cells. At concentrations > or = 10(-9) M RK-1 decreased enterocyte volume in a dose-dependent manner through a pathway that requires extracellular calcium and is inhibited by niguldipine, a dihydropyridine-sensitive "L"-type Ca(2+)-channel blocker. In other assay systems for corticostatin-defensins, such as the inhibition of adrenocorticotropin-stimulated steroidogenesis, or cell lysis, RK-1 was inactive or only weakly active. These results demonstrate the existence of a novel system of biologically active peptides in the kidney represented by RK-1 which is antimicrobial and can activate epithelial ion channels in vitro.