Growth factor PDGF-BB stimulates cultured cardiomyocytes to synthesize the extracellular matrix component hyaluronan

Background

Hyaluronan (HA) is a glycosaminoglycan located in the interstitial space which is essential for both structural and cell regulatory functions in connective tissue. We have previously shown that HA synthesis is up-regulated in a rat model of experimental cardiac hypertrophy and that cardiac tissue utilizes two different HA synthases in the hypertrophic process. Cardiomyocytes and fibroblasts are two major cell types in heart tissue. The fibroblasts are known to produce HA, but it has been unclear if cardiomyocytes share the same feature, and whether or not the different HA synthases are activated in the different cell types.

Methodology/Principal Findings

This study shows, for the first time that cardiomyocytes can produce HA. Cardiomyocytes (HL-1) and fibroblasts (NIH 3T3) were cultivated in absence or presence of the growth factors FGF2, PDGF-BB and TGFB2. HA concentration was quantified by ELISA, and the size of HA was estimated using dynamic light scattering. Cardiomyocytes synthesized HA but only when stimulated by PDGF-BB, whereas fibroblasts synthesized HA without addition of growth factors as well as when stimulated by any of the three growth factors. When fibroblasts were stimulated by the growth factors, reverse dose dependence was observed, where the highest dose induced the least amount of HA. With the exception of TGFB2, a trend of reverse dose dependence of HA size was also observed.

Conclusions/Significance

Co-cultivation of cardiomyocytes and fibroblasts (80%/20%) increased HA concentration far more that can be explained by HA synthesis by the two cell types separately, revealing a crosstalk between cardiomyocytes and fibroblasts that induces HA synthesis. We conclude that dynamic changes of the myocardium, such as in cardiac hypertrophy, do not depend on the cardiomyocyte alone, but are achieved when both cardiomyocytes and fibroblasts are present.     

Inducible nitric oxide synthase is involved in acid-induced gastric hyperemia in rats and mice

The role of different isoforms of nitric oxide synthase (NOS) in the gastric mucosal hyperemia, induced by 155 mM luminal hydrochloric acid (pH approximately 0.8) without a barrier breaker, was investigated. Rats were anesthetized with Inactin (120 mg/kg ip), and mice were anesthetized with Forene (2.2% in 40% oxygen gas at 150 ml/min); the gastric mucosa was exteriorized. Gastric mucosal blood flow was measured with laser-Doppler flowmetry (LDF) in rats treated with Nomega-nitro-l-arginine (l-NNA; unspecific NOS inhibitor), l-N6-(1-iminoethyl)lysine [l-NIL; inducible (i) NOS inhibitor], or S-methyl-l-thiocitrulline [SMTC; neuronal (n) NOS inhibitor], 10 mg/kg, followed by 3 mg. kg-1. h-1 iv, in iNOS-deficient (-/-) and nNOS(-/-) mice. mRNA was isolated from the gastric mucosa in iNOS(-/-) and wild-type (wt) mice, and real-time RT-PCR was performed. The effect of 155 mM acid on gastric mucosal permeability was determined by measuring the clearance of 51Cr-EDTA from blood to lumen. LDF increased by 48 +/- 13% during 155 mM HCl luminally, an increase that was abolished by l-NNA, SMTC, or l-NIL. In iNOS wt mice, LDF increased by 33 +/- 8% during luminal acid. The blood flow increase was attenuated substantially in iNOS(-/-) mice. RT-PCR revealed iNOS mRNA expression in the gastric mucosa in the iNOS wt groups. The blood flow increase in response to acid was not abolished in nNOS(-/-) mice (nNOS-sufficient mice, 39 +/- 18%; heterozygous mice, 25 +/- 19%; -/- mice, 19 +/- 7%). Mucosal permeability was transiently increased during 155 mM HCl. The results suggest that iNOS is constitutively expressed in the gastric mucosa and is involved in acid-induced hyperemia, suggesting a novel role for iNOS in gastric mucosal protection.     

Canine sulfotransferase SULT1A1: molecular cloning,expression, and characterization

Sulfotransferases (SULTs) are involved in detoxification and activation of various endogenous and exogenous compounds including important drugs and hormones. SULT1A, the phenol-SULT subfamily, is the most prominent subfamily in xenobiotic metabolism and has been found in several species, e.g., human, rat, and mouse. We have cloned a phenol-sulfating phenol SULT from dog (cSULT1A1) and expressed it in Escherichia coli for characterization. cSULT1A1 showed 85.8, 82.7, 76.3, and 73.6% identities to human P-PST, human M-PST, rat PST-1, and mouse STp1, respectively. It consists of 295 amino acids, which is in agreement with the human ortholog and sulfate substrates typical for the SULT1A family, i.e., p-nitrophenol (PNP), alpha-naphthol, and dopamine. The K(m) for PNP was found to be within the nanomolar range. It also sulfates minoxidil and beta-estradiol but not dehydroepiandrosterone. Western blot analysis indicated that this newly cloned enzyme was found to be ubiquitously expressed in canine tissues with highest expression in male and female liver.     

Purification and characterization of two forms of a low-affinity Ca2+-ATPase from erythrocyte membranes

A low-affinity Ca²⁺-ATPase from erythrocyte membranes has been purified by agarose suspension electrophoresis and polyacrylamide gel electrophoresis in the absence of detergents. For maximal activity a calcium concentration above 10 mM is required. The activity is independent of magnesium. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for ATP is about 60 μM. The enzyme appears in two forms (A and B) with similar amino acid composition. The specific activity of A is higher than that of B. Gel electrophoresis in SDS of A gives a pattern consisting of two bands. B gives the same pattern; the only difference between the patterns is the ratio of the amounts of protein in the bands. The apparent molecular weight of the proteins in the two SDS bands has been estimated at 23 000 and 21 000, respectively. The results obtained can be explained by assuming that the two proteins corresponding to the two bands obtained in SDS electrophoresis have a similar structure and can associate to complexes A and B. We have also shown that electrophoretic and chromatographic supporting media can induce aggregation of (membrane) proteins. Artificial complexes can thus be formed and cause misinterpretation of the data obtained. This may be the reason why some authors have speculated that Ca²⁺-ATPase is active only in complex with other proteins such as spectrin and actin.     

Short- and long-term effects of dimethyl sulfoxide on mouse pancreatic islet B-cell function in vitro

Dimethylsulfoxide has been used as a cryoprotectant for the endocrine pancreas. To explore possible harmful effects of Me₂SO influenced neither glucose-stimulated insulin release, nor islet glucose oxidation. On the other hand, 1 M but not 0.25 M Me₂SO decreased glucose-stimulated (pro)insulin and total protein biosynthesis in acute experiments. In islet culture experiments with Me₂SO-supplemented culture media there was no obvious effects on glucose-stimulated (pro)insulin biosynthesis. It is suggested that Me₂SO in the range 0.01–0.5 M does not affect islet B-cell function in vitro.