The ATP/DNA ratio is a better indicator of islet cell viability than the ADP/ATP ratio.

Real-time, accurate assessment of islet viability is critical for avoiding transplantation of nontherapeutic preparations. Measurements of the intracellular ADP/ATP ratio have been recently proposed as useful prospective estimates of islet cell viability and potency. However, dead cells may be rapidly depleted of both ATP and ADP, which would render the ratio incapable of accounting for dead cells. Since the DNA of dead cells is expected to remain stable over prolonged periods of time (days), we hypothesized that use of the ATP/DNA ratio would take into account dead cells and may be a better indicator of islet cell viability than the ADP/ATP ratio. We tested this hypothesis using mixtures of healthy and lethally heat-treated (HT) rat insulinoma cells and human islets. Measurements of ATP/DNA and ADP/ATP from the known mixtures of healthy and HT cells and islets were used to evaluate how well these parameters correlated with viability. The results indicated that ATP and ADP were rapidly (within 1 hour) depleted in HT cells. The fraction of HT cells in a mixture correlated linearly with the ATP/DNA ratio, whereas the ADP/ADP ratio was highly scattered, remaining effectively unchanged. Despite similar limitations in both ADP/ADP and ATP/DNA ratios, in that ATP levels may fluctuate significantly and reversibly with metabolic stress, the results indicated that ATP/DNA was a better measure of islet viability than the ADP/ATP ratio.

Human islet oxygen consumption rate and DNA measurements predict diabetes reversal in nude mice.

There is a need for simple, quantitative and prospective assays for islet quality assessment that are predictive of islet transplantation outcome. The current state-of-the-art athymic nude mouse bioassay is costly, technically challenging and retrospective. In this study, we report on the ability of 2 parameters characterizing human islet quality: (1) oxygen consumption rate (OCR), a measure of viable volume; and (2) OCR/DNA, a measure of fractional viability, to predict diabetes reversal in nude mice. Results demonstrate that the probability for diabetes reversal increases as the graft's OCR/DNA and total OCR increase. For a given transplanted OCR dose, diabetes reversal is strongly dependent on OCR/DNA. The OCR and OCR/DNA (the 'OCR test') data exhibit 89% sensitivity and 77% specificity in predicting diabetes reversal in nude mice (n = 86). We conclude that the prospective OCR test can effectively replace the retrospective athymic nude mouse bioassay in assessing human islet quality prior to islet transplantation.

Absolute concentrations of mRNA for type I and type VI collagen in the canine meniscus in normal and ACL-deficient knee joints obtained by RNase protection assay.

Relatively little is known about the cellular and molecular responses of the knee joint meniscus to joint injury, despite the functional importance of the tissue. We investigated how meniscus cells respond to joint injury in the early stages of post-traumatic osteoarthritis by characterizing the changes in matrix gene expression in menisci at 3 and 12 weeks post-surgery in dogs in which the anterior cruciate ligament (ACL) in one joint was transected and the other unoperated joint served as a control. Changes in the total RNA and DNA concentrations of the menisci were determined. Absolute concentrations of the mRNA of the COL1A1 gene of type 1 collagen, the major fibrillar collagen of the meniscus, and the COL6A3 gene of type VI collagen, a major repair molecule, were determined by quantitative ribonuclease (RNase) protection assay. The concentration of total RNA in medial and lateral menisci increased from 40 to 60 microg RNA/g wet wt in unoperated, control joints to 200-350 microg RNA/g wet wt in ACL-deficient joints. No significant changes were detected in the concentration of DNA (900-1200 microg DNA/g wet wt). Low concentrations of COL1A1 (2-3 pmol mRNA/g DNA) and COL6A3 (0.3-0.6 pmol mRNA/g DNA) mRNA transcripts were measured in normal menisci. ACL-deficiency induced a 20-38 fold increase in COL1A1 and COL6A3 mRNA concentration at 3 weeks, and an 11-19 fold increase at 12 weeks post-surgery. In general, the increase in COL1A1 and COL6A3 mRNA concentrations was greater in medial menisci than in lateral menisci. These results demonstrate that the menisci initiate a vigorous biosynthetic response to transection of the ACL.

Smad7 is induced by CD40 and protects WEHI 231 B-lymphocytes from transforming growth factor-beta -induced growth inhibition and apoptosis.

Transforming growth factor-beta (TGF-beta) is a potent inducer of apoptosis in B-lymphocytes and is essential for immune regulation and maintenance of self-tolerance. Here we show that concomitant signaling through CD40 sustains proliferation and rescues the premature B cell line WEHI 231 from both TGF-beta-induced and anti-IgM-induced apoptosis. The anti-apoptotic effect of CD40 is associated with the transcriptional activation of the inhibitory Smad7 protein. The transactivation of Smad7 by CD40 is NFkappaB-dependent in that pharmacological inhibitors of this pathway, N-tosyl-l-phenylalanine chloromethyl ketone and pyrrolidine dithiocarbamate, abrogate CD40-induced Smad7 expression. Ectopic overexpression of Smad7 inhibited Smad2 activation, TGF-beta-mediated growth inhibition, and apoptosis in WEHI 231 cells. Consistent with this result, dominant negative interference with Smad2 and Smad3 function also inhibited TGF-beta-induced apoptosis. The inhibitory effects of Smad7 overexpression were specific to TGF-beta-induced apoptosis and were without effect on anti-IgM-induced cell death. These results suggest a mechanism of suppression of TGF-beta-induced apoptosis by CD40, mediated through activation of NF-kappaB and, consequently, induction of Smad7 expression.

Matrix protein mRNA levels in canine meniscus cells in vitro.

The resident cells of the meniscus synthesize a fibrocartilaginous extracellular matrix in vivo composed predominantly of type I collagen fibers. To increase our understanding of matrix biosynthesis by meniscus cells in vitro, we examined matrix protein mRNA levels in cultured meniscus cells isolated from skeletally mature dogs. The mRNA levels of five matrix protein genes (COL1A1, COL2A1, aggrecan, COL6A1, and fibronectin) were measured in meniscus cells by Northern blotting and compared with those of patellar tendon fibroblasts and femoral articular cartilage chondrocytes. In freshly isolated cells (Day 0 cells), COL1A1, COL2A1, and aggrecan mRNA levels were low or undetectable in both meniscus cells and tendon fibroblasts. In intact meniscus tissue, COL1A1 mRNA levels were also low or undetectable. COL2A1 and aggrecan mRNA transcripts were readily observed, however, in Day 0 articular chondrocytes. The levels of expression of COL6A1 and fibronectin mRNA transcripts in Day 0 meniscus cells were intermediate between higher articular chondrocyte levels and lower tendon fibroblast levels. After 1 week in monolayer culture (Day 7 cells), meniscus cells expressed readily detectable levels of COL1A1 mRNA transcripts, similar to that observed for cultured tendon fibroblasts. COL1A1 mRNA transcripts were either not detected or detected at very low levels in monolayer cultures of articular chondrocytes. COL2A1 and aggrecan mRNA transcripts were readily detected in cultured articular chondrocytes but not in meniscus cells or in tendon fibroblasts. All three types of cells continued to express COL6A1 and fibronectin mRNA transcripts after 1 week in culture. These results demonstrate that the patterns of expression of COL1A1 and COL2A1 mRNA transcripts by meniscus cells are similar to those of tendon fibroblasts and dissimilar to those of articular chondrocytes both in freshly isolated cells and in monolayer cultured cells. This mRNA expression pattern supports the idea that monolayer culture of meniscus cells results in the expression of a predominantly fibroblastic phenotype.

Cloning and tissue expression of the rat RAB 3C GTP-binding protein.

A cDNA clone which encodes the low molecular weight GTP-binding protein rab 3C was isolated from a rat PC12 pheochromocytoma library. The 1.0 kb clone contains the entire coding region (681 bp), as well as 5' (89 bp) and 3' (230 bp) untranslated sequences. The predicted amino acid sequence of the rat rab 3C clone is 98% identical to the bovine rab 3C sequence and 85% identical to the rat rab 3A sequence. Northern blot analyses using probes containing coding and noncoding sequences of the rat rab 3C clone hybridized to a 9.5 kb transcript in brain, adrenal gland, and pituitary RNA pools.

Brefeldin A defines distinct pathways for atrial natriuretic factor secretion in neonatal rat atrial and ventricular myocytes.

The intracellular pathways for basal atrial natriuretic factor (ANF) secretion from the heart and their correlation with ANF processing to the active form were characterized in cultured neonatal rat atrial and ventricular myocytes. Brefeldin A, a fungal antimetabolite that blocks transport of newly synthesized proteins from the endoplasmic reticulum, was used to inhibit nascent protein trafficking. Thus, release of newly synthesized hormone was blocked, but release of stored hormone was unaffected. Whereas brefeldin A inhibited basal ventricular ANF release to 10% of the control value, basal ANF release from atrial cells was enhanced. Furthermore, basal atrial ANF secretion was inhibited by agents preventing myocyte depolarization, Ca2+ influx, release of Ca2+ from intracellular stores, or activation of protein kinase C, whereas ventricular ANF secretion was unaffected by these agents. Brefeldin A did not alter maturational processing of pro-ANF to ANF-(99-126) in either atrial or ventricular cultures. These findings indicate that (1) basal secretion of ANF from ventricular cells relies largely on newly synthesized hormone and is probably constitutive, (2) basal secretion of ANF from atrial cells is independent of transport of newly synthesized protein and occurs via a regulated pathway controlled at least in part by signaling changes associated with myocyte beating, and (3) processing of pro-ANF occurs either with constitutive or regulated secretion of hormone, which may indicate multiple cellular locations for the processing enzyme.

Isolation of cDNA clones and tissue expression of rat ral A and ral B GTP-binding proteins.

cDNA clones encoding the low molecular weight GTP-binding proteins ral A (951 bp) and ral B (2073 bp), including the entire coding region (618 bp), were isolated from a rat PC12 pheochromocytoma library. Northern analyses demonstrated that both ral A and ral B are widely expressed in rat tissues. Two ral A transcripts of 1.1 and 2.9 kb were observed in most tissues in varying proportions. The 1.1 kb ral A band of testes was further shown to be composed of two closely migrating species. In contrast to these findings, a single ral B transcript of 2.3 kb was detected in most tissues. Steady-state levels of ral A transcripts appear greater than ral B. Quantitatively, the testes exhibited the highest ral A and ral B mRNA levels, with lower levels observed in the brain, adrenal and pituitary glands, kidney and ovary. Ral mRNA levels were lowest in muscle tissue, particularly skeletal muscle.

Detection of low molecular weight GTP-binding proteins associated with rat atrial secretory granules.

Recent studies suggest that low molecular weight (LMW) GTP-binding proteins are involved in the vesicular transport of proteins along the secretory pathway. In order to determine whether LMW GTP-binding proteins are potentially involved in the regulated secretion of atrial natriuretic factor (ANF) from the atrial myocyte, rat atrial secretory granules were purified and the LMW GTP-binding proteins associated with these granules were detected in [alpha-32P]GTP-blotting experiments. Three LMW GTP-binding proteins, displaying molecular weights of 23,000, 25,000 and 29,000, were usually observed in atrial secretory granule preparations. The 29 kDa GTP-binding protein (G29), however, was typically the predominantly labelled band. The binding of [alpha-32P]GTP to G29 was guanine nucleotide specific and magnesium-dependent. Trypsin treatment of intact secretory granules markedly reduced the binding of [alpha-32P]GTP to G29. G29 is tightly associated with the granule membrane, as evidenced by its resistance to solubilization by high ionic strength buffers but its sensitivity to solubilization with sodium cholate. When secretory granule preparations of rat anterior pituitary glands were examined for the presence of LMW GTP-binding proteins, a different pattern of LMW GTP-binding proteins was observed which did not include a predominantly labelled 29 kDa protein. The results of this study indicate that: (1) LMW GTP-binding proteins are present on atrial secretory granules and may therefore be involved in the regulated secretion of ANF and (2) tissue-specific differences exist between the LMW GTP-binding proteins associated with secretory granules.

Phosphorylation state of pro-atrial natriuretic factor in rat atrial secretory granules.

Recent studies have demonstrated that phosphorylation of atrial natriuretic factor (ANF) (99-126) in vitro modulates the bioactivity of this hormone. The potential physiological relevance of this observation was revealed in latter studies showing that endogenous proANF can be 32PO4-biosynthetically labeled by primary cultured atrial myocytes and by atrial appendage explants. The site and extent of proANF phosphorylation were different, however, in these two model systems. Whereas proANF extracted from atrial explants was phosphorylated on the bioactive, carboxy (C)-terminal portion of the molecule [ANF(99-126)], cultured atrial myocytes phosphorylated proANF on the amino (N)-terminal portion of the prohormone molecule [ANF(1-98)]. It was the goal of this study, therefore, to determine whether the bioactive region of proANF, ANF(99-126), is phosphorylated in vivo. ProANF was obtained by acid extraction of isolated rat atrial secretory granules followed by purification using reverse phase-HPLC. Analysis of purified 125I-labeled proANF by isoelectric focusing (IEF) revealed two bands with isoelectric points of 5.3 and 5.0. The more acidic band comigrated on IEF gels with 32PO4-biosynthetically labeled proANF obtained from primary cultures of atrial myocytes, suggesting that this species of proANF represented endogenously phosphorylated proANF. The more acidic band accounted for only 15-25% of the total proANF found in the mature atrial secretory granule. The phosphorylation state of ANF(99-126) produced by thrombin cleavage of secretory granule proANF was examined using three complementary methods: 1) cation-exchange HPLC, 2) amino-terminal amino acid sequence analysis and 3) anti-ANF(99-105) antibody immunoreactivity. Evidence from these three independent approaches indicated that proANF is not phosphorylated on the C-terminal portion of the molecule in vivo. Therefore, phosphorylation is not a physiological regulator of ANF(99-126) bioactivity.

Biochemical studies of soluble atrial natriuretic peptide (ANP) receptors from rat olfactory bulb and vascular smooth muscle cells.

1. Aim. The biochemical characteristics of atrial natriuretic peptide receptors (ANP-R) derived from rat vascular smooth muscle (A-10 cell line) and central nervous system (CNS; olfactory bulb) tissue were compared. 2. Method and Results. ANP-Rs from each source were solubilized with 40 to 65% efficiency utilizing the nonionic detergent Lubrol-PX. Upon solubilization, the ANP-R from each source maintained the ability to bind 125I-ANP (99-126) with a high affinity; Scatchard analysis indicated that the VSMC ANP-R displayed a Kd for the radioligand of approximately 10 pM, whereas the olfactory receptor possessed a Kd of about 165 pM. The Bmax values for the soluble VSMC and olfactory ANP-Rs were 285 and 30 fmol/mg protein, respectively. Competition binding studies indicated that the VSMC ANP-R bound ANP(99-126), ANP(103-126), and ANP(103-123) with similar affinities, whereas the olfactory ANP-R was much more sensitive to changes in the COOH-terminal structure of the competing peptide. The soluble ANP-Rs from VSMC and olfactory were chromatographically indistinguishable on phenyl-, DEAE-, and wheat germ agglutinin-agarose columns. However, the ANP-Rs could be distinguished using GTP-agarose; the olfactory ANP-R was capable of binding to the resin, whereas the VSMC ANP-R was not. 3. Conclusions. Coupled with other studies, these data suggest that the A10 VSMC ANP-R observed in this study may not be coupled to guanylate cyclase and may represent a receptor serving a clearance function, whereas a significant proportion of the olfactory CNS ANP-R appears to be associated with GTP-binding proteins, likely particulate guanylate cyclase, and probably represents a coupled form of the receptor.

Specificity of serine proteases for cleavage sites on proatrial natriuretic factor.

An immunological approach was used to investigate the specificity of protease cleavage sites on proANF. Cleavage of 35S-cysteine biosynthetically-labeled proANF by whole serum, thrombin and kallikrein was examined. Reaction products were immunoprecipitated with two antibodies directed to different epitopes: a previously characterized antibody directed toward the carboxy-terminus of ANF103-126, which cross reacts with proANF, ANF99-126 and ANF103-126, and a newly prepared antisera to synthetic ANF99-105, which uniquely recognizes ANF99-126, but not proANF or ANF103-126. With increasing time of incubation with rat serum, proANF is sequentially cleaved at the C-terminus of a monobasic Pro-Arg dipeptide sequence to form ANF99-126, and then at the C-terminus of a dibasic Arg-Arg dipeptide sequence to yield ANF103-126. This cleavage activity of serum is blocked by leupeptin (40 micrograms/ml), but not by hirudin (100 nM), a specific inhibitor of thrombin, or by aprotinin (200 KIU/ml), a kallikrein inhibitor. When 100-fold purified serum cleavage enzyme was used in place of crude serum, similar results were obtained. Thrombin cleaves proANF only at the monobasic site to produce ANF99-126 while kallikrein cleaves only at the dibasic site to produce ANF103-126. As expected, the generation of these cleavage products can be inhibited by hirudin or aprotinin respectively. These data indicate that the substrate specificity of the serum cleavage activity is broader than that of thrombin or kallikrein, and that cleavage of proANF by serum proteases may be influenced by conformational restraints. The methods developed here should help in the future characterization of the physiological proANF cleaving enzyme.

Cellular processing of pro-atrial natriuretic factor (pro-ANF): studies using an antiserum that selectively binds ANF-(99-126) after its cleavage from pro-ANF.

Atrial natriuretic factor (ANF) is stored in atrial myocytes as a 15-17K prohormone, but circulates in plasma as a 3K, carboxy (C)-terminal fragment of the prohormone. The tissue location at which the cleavage of pro-ANF to its hormonal form occurs is unknown. In the present study, an immunological approach was taken to address this question. A polyclonal antiserum was generated which recognizes the hormonal form of ANF [ANF-(99-126)] only after its cleavage from the prohormone. This was accomplished by immunizing rabbits with a synthetic peptide corresponding to the seven amino (N)-terminal residues of ANF-(99-126) coupled to carrier protein via a C-terminal cysteine. This antiserum, anti-ANF-(99-105), demonstrated high affinity for ANF-(99-126) (IC50 = 170 pM), but displayed 100-fold less affinity for recombinant pro-ANF [ANF-(2-126)]. The N-terminal specificity of anti-ANF-(99-105) was evident by its failure to bind ANF-(103-126) at concentrations up to 100 nM. The specificity of anti-ANF-(99-105) for the hormonal form of ANF was examined by using thrombin to cleave pro-ANF and testing for the generation of anti-ANF-(99-105) immunoreactivity. Cleavage of atrial pro-ANF or 35S biosynthetically-labeled pro-ANF resulted in the production of immunoreactive material from the prohormone, whereas pro-ANF itself demonstrated no cross-reactivity with anti-ANF-(99-105). Anti-ANF-(99-105) could also recognize ANF released from the isolated perfused rat heart. When anti-ANF-(99-105) was used in immunohistochemical studies of rat atrial myocardium, no staining was observed in unfixed frozen sections. This suggests that proteolytic processing of pro-ANF is not an intracardiocytic event.

Characterization and purification of a protease in serum that cleaves proatrial natriuretic factor (ProANF) to its circulating forms.

Atrial natriuretic factor (ANF) is synthesized and stored in atrial cardiocytes as a 17-kilodalton (kDa), 126 amino acid polypeptide, proANF, but circulates as smaller, 24 and 28 amino acid peptide fragments of the carboxy terminus of proANF. It has previously been shown that proANF is secreted intact from cultured atrial cardiocytes and can be cleaved by a serum protease to smaller, 3-kDa peptides believed to be the circulating forms. This report describes the purification and characterization of this proANF-cleaving protease from rat serum. The cleavages both of 35S-labeled proANF derived from rat atrial cell cultures, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/autoradiography, and of a synthetic p-nitroanilide-containing substrate were used as assays for the detection of enzyme activity. ProANF-cleaving activity was found in rat serum, with no such activity detectable in rat plasma. Cleavage in serum was not dependent on the presence of platelets or other cellular elements. Complete inhibition of proANF cleavage was obtained with the protease inhibitors benzamidine, leupeptin, phenylmethanesulfonyl fluoride, and diisopropyl fluorophosphate (DFP) but not with aprotinin, soybean trypsin inhibitor, pepstatin, or hirudin. Unlike the vitamin K dependent plasma proteins, the proANF-cleaving protease did not adsorb to barium sulfate. With the sequential application of ion-exchange, hydroxylapatite, lectin affinity, and gel filtration chromatography, a 5000-6000-fold purification of the enzyme from rat serum was achieved. Fractionation of either whole serum or the purified enzyme by gel filtration chromatography revealed a single peak of activity corresponding to a protein with a Stokes radius of 45 A.(ABSTRACT TRUNCATED AT 250 WORDS)

Cross-linking of atrial natriuretic peptide to binding sites in rat olfactory bulb membranes.

Binding sites for 125I-atrial natriuretic peptide (ANP)2 in rat olfactory bulb membranes have been studied using pharmacological and biochemical methods. Various unlabeled ANP-related peptides were tested for the ability to inhibit the binding of the radioligand in membrane binding assays. ANP(92-126) and ANP(99-126) were the most potent inhibitors tested, both exhibiting an IC50 value of 0.40 nM. ANP(103-126) and ANP(103-123) were 3 and 70 times less potent, respectively. ANP(111-126) was unable to inhibit the binding of the radioligand at a concentration of 1 microM. Several peptides unrelated to ANP were unable to inhibit the binding of the radioligand to rat olfactory bulb membranes. Membranes labeled with 125I-ANP were incubated with cross-linking agents and subjected to SDS-PAGE followed by autoradiography. A band possessing an apparent molecular mass of 116 kDa was identified. The labeling of this band was progressively decreased by increasing concentrations of unlabeled ANP(99-126) (IC50 = 0.6 nM) and by several other ANP-related peptides at nanomolar concentrations. For comparison purposes, ANP binding sites in rat aorta membranes were labeled with 125I-ANP and cross-linked using identical techniques. Three bands possessing molecular masses of 120, 72, and 62 kDa were identified. These results indicate that the ANP binding site in rat olfactory bulb membranes displays pharmacological and biochemical properties similar to peripheral ANP receptors.

Autoradiographic localization and characterization of atrial natriuretic peptide binding sites in the rat central nervous system and adrenal gland.

Atrial natriuretic peptides (ANP) have recently been identified in both heart and CNS. These peptides possess potent natriuretic, diuretic, and vasorelaxant activities, and are all apparently derived from a single prohormone. Specific ANP binding sites have been characterized in the adrenal zona glomerulosa and kidney cortex, and one study reported ANP binding sites in the CNS. However, a detailed examination of the localization of ANP binding sites throughout the brain has not been reported. In this study, quantitative autoradiography was employed to examine the distribution of ANP receptors in the rat CNS. The binding of (3-125I-iodotyrosyl28) rat ANP-28 to binding sites in the rat CNS was saturable, specific for ANP-related peptides, and displayed high affinity (Kd = 600 pM). When the relative concentrations of ANP binding sites were determined throughout the rat brain, the highest levels of ANP binding were localized to the circumventricular organs, including the area postrema and subfornical organ, and the olfactory apparatus. Moderate levels of ANP binding sites were present throughout the midbrain and brain stem, while low levels were found in the forebrain, diencephalon, basal ganglia, cortex, and cerebellum. The presence of ANP binding sites in the subfornical organ and the area postrema, regions considered to be outside the blood-brain barrier, suggests that peripheral ANP levels may regulate some aspects of CNS control of salt and water balance. The possible functions of ANP binding sites in other regions of the rat brain are not known, but, like many other peptides, ANP may act as a neurotransmitter or neuromodulator at these loci.

Molecular forms of immunoactive atrial natriuretic peptide in the rat hypothalamus and atrium.

Acid extracts of rat hypothalamus and atrium were prepared by a procedure previously shown to minimize proteolytic degradation of peptides. The majority of the immunoactive material in the atrial extracts had a molecular weight of approximately 9,000 to 15,000 daltons, while that in the hypothalamic extracts had a molecular weight of about 1,500 to 1,800 daltons. The major molecular weight forms of atrial natriuretic peptide from each extract were further distinguishable when analyzed by RP-HPLC. These results suggest that small peptides such as atriopeptins I, II, and III, may not be authentic post-translational processing products in the atrium, and that the hypothalamus and atrium may differentially cleave pro-atrial natriuretic peptide to form tissue-specific products.