Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purified from the breast muscles of 3-week-old chickens and used to raise a specific antiserum in rabbits. This antiserum was coupled to an in vitro translation assay to monitor the purification of GAPDH mRNA. RNA was isolated from identical breast muscles and consecutively fractionated with several techniques to yield a preparation of GAPDH mRNA which was at least 50% pure. Double-stranded cDNA was made against this purified RNA, inserted into pBR322, and used to transform Escherichia coli. Recombinants were screened by colony filter hybridization with a cDNA probe made against the purified RNA. The hybridization-positive clone with the largest insert, pGAD-28, was then characterized by using pGAD-28-cellulose to select complementary RNA from total poly(A) RNA and then translating the hybridization-selected RNA in vitro. The single translation product was shown to be GAPDH by (1) comigration with pure GAPDH on sodium dodecyl sulfate-polyacrylamide gels, (2) precipitation with specific anti-GAPDH antiserum, (3) cyanylation fingerprinting, and (4) AMP-agarose affinity chromatography. pGAD-28 was mapped with several restriction enzymes and then sequenced by the method of Maxam and Gilbert [Maxam, A. M., & Gilbert, W. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 560]. The 1261-nucleotide insert was found to contain 29 nucleotides of noncoding sequence at the 5' end, the entire coding region, and 230 nucleotides of the 3'-noncoding region including a poly(A) addition signal (AATAAA) and the first five residues of the poly(A) tail.

A peptide corresponding to the epidermal growth factor homology domain of ␤-heregulin stimulated autophosphorylation of the heregulin receptors erbB2 and erbB3 in Schwann cells and activation of the mitogen-activated protein (MAP) kinases ERK1 and ERK2. Heregulin-dependent activation of PAK65, a component of the stress-activated signaling pathway, ribosomal S6 kinase, and a cyclic AMP (cAMP) response element binding protein (CREB) kinase, identified as p95 RSK2, was also observed. Receptor phosphorylation and activation of these kinases in response to heregulin occurred in the absence of forskolin stimulation and were not augmented in cells treated with forskolin, a direct activator of adenylyl cyclase. Schwann cell proliferation in response to heregulin was observed only when the cells were also exposed to an agent that elevates cAMP levels. In the absence of heregulin, elevation of cAMP levels failed to stimulate Schwann cell proliferation. Forskolin significantly enhanced heregulin-stimulated expression of cyclin D and phosphorylation of the retinoblastoma gene product. In cells treated with both heregulin and forskolin there was a sustained accumulation of phospho-CREB, which was not observed in cells treated with either agent alone. Heregulin and forskolin synergistically activated transcription of a cyclin D promoter construct. These results demonstrate that heregulin-stimulated activation of MAP kinase is not sufficient to induce maximal Schwann cell proliferation. Expression of critical cell cycle regulatory proteins and cell division require activation of both heregulin and cAMP-dependent processes.Myelination of axons by Schwann cells is critical for the proper functioning of the peripheral nervous system. The correct ratio of Schwann cells to axons is achieved during development through a combination of Schwann cell proliferation (26) and programmed cell death (29). Studies with primary cultures of Schwann cells and embryonic sensory neurons have shown that molecular signals that stimulate Schwann cell proliferation are associated with axonal membranes (24,27,35).Several lines of evidence suggest that the axonal Schwann cell mitogen is a member of the heregulin family of growth factors (5,9,17,21). A common structural feature of heregulins is a cysteine-rich domain of approximately 50 amino acids that is homologous to the active domain of epidermal growth factor (EGF) (18). Heregulins stimulate cell proliferation by binding to and activating transmembrane receptor tyrosine kinases with homology to the EGF receptor, called erbB2, erbB3, and erbB4 (10, 25). A synthetic peptide corresponding to the heregulin EGF homology domain is sufficient to mediate binding to erbB receptors (2). Ligand-dependent activation of erbB receptors leads to activation of the mitogen-activated protein (MAP) kinase pathway, which is critical for cell division in many cell types (22).Schwann cell proliferation can also be stimulated by other polypeptide growth factors (6), including basic fibroblast growth factor and platelet-d...

There is increasing evidence that the renin-angiotensin system (RAS) modulates cardiovascular function through both blood-borne and tissue-derived components. The existence of a local RAS has been proposed in the heart based on biochemical and molecular biological studies that identify angiotensinogen and renin. We conducted the present study to determine the chamber localization of angiotensinogen and renin mRNA in neonatal rat heart and whether these components could be identified in cultured cardiomyocytes and fibroblasts obtained from neonatal rat heart. Experiments using polymerase chain reaction (PCR) indicated that whole hearts obtained from neonatal rats contained both angiotensinogen and renin mRNA. With the use of radiolabeled cDNA probes and in situ hybridization, angiotensinogen and renin transcripts were localized both in the atria and ventricles of neonatal rat hearts. Relative signal strengths for angiotensinogen were highest in the left and right ventricles. In contrast, renin signal strength was overall much lower and preferentially localized in the left ventricle. To investigate the cellular source of angiotensinogen and renin, cultured neonatal heart cardiomyocytes and ventricular fibroblasts were screened for angiotensinogen and renin messenger RNA and protein using PCR and indirect immunofluorescent staining, respectively. These experiments demonstrated that both cell types produce transcripts and the respective translation products for angiotensinogen and renin. These data suggest that the site of angiotensin II synthesis can occur at the level of the individual cardiomyocyte and fibroblast, where it may serve to directly and/or indirectly regulate cardiac rate, force, growth, and development in the neonate.

We described the construction of an alpha-actin complementary deoxyribonucleic acid (cDNA) clone, pAC269 [Schwartz, R. J., Haron, J. A., Rothblum, K. N., & Dugaiczyk, A. (1980) Biochemistry 19, 5883], that was used as a hybridization probe in the current investigation to examine the induction of actin messenger ribonucleic acid (mRNA) during myogenesis. A Tm difference of 10-13 degrees C between skeletal muscle alpha-actin and nonmuscle beta- and gamma-actin mRNAs and pAC269 allowed us to establish the highly stringent hybridization conditions necessary to measure separately the content of alpha-actin mRNA and beta- and gamma-actin mRNA during muscle development in culture. We observed low levels of alpha-actin mRNA (approximately 130 molecules/cell) in replicating prefusion myoblasts. The vast majority of actin mRNA (2000 molecules/cell) present at this stage was accounted for by beta- and gamma-actin mRNA. Beginning at myoblast fusion, alpha-actin mRNA accumulated and within 30 h reached a level 270-fold greater than that observed in the undifferentiated state. At 95 h in culture when myotube formation was completed, alpha-actin content was at its peak (36 000 molecules/nucleus). Conversely, beta- and gamma-actin mRNA content began to decline at the beginning of fusion, and by the end of myotube formation beta- and gamma-actin mRNAs were undetectable by our techniques. A rapid depression of alpha-actin mRNA levels was observed after 95 h in the absence of cell death. At 6 days after the initiation of myotube formation, the content of alpha-actin mRNA was reduced by 80% in comparison of peak values and remained at that level. The switching of actin mRNA species was inhibited in myoblasts treated with bdU. The accumulation of alpha-actin mRNA and the disappearance of beta- and gamma-actin mRNA were observed following the reversal of the bdU block and coincident with the onset of myoblast fusion. We found that the expression of actin genes within the actin multigene family is switched in myogenesis through a strict developmental pattern.

Angiotensin II (ANG II) is a stimulus for positive chronotropic and inotropic effects, protein synthesis, and hypertrophic growth in cardiac tissue. These short- and long-term effects of ANG II are mediated through specific plasma membrane receptors. Indirect evidence suggests that ANG II synthesized in the myocardium may be important in regulating cardiac function. The cell types in the myocardium that produce components of the renin-angiotensin system have not been determined. In this study, we evaluated whether cultured cardiomyocytes and fibroblasts obtained from ventricles of neonatal rat hearts were capable of synthesizing ANG I and II. Both cardiomyocytes and fibroblasts were found to have immunofluorescent staining for ANG I, ANG II, and angiotensin-converting enzyme (ACE). The amounts of ANG I and II in cell extracts and conditioned media obtained from cardiomyocytes and fibroblasts were quantified by radioimmunoassay. The amounts of ANG I and II detected in cardiomyocyte cultures (1.48 x 10(6) cells/dish) were 32.2 +/- 16.2 (n = 4) and 6.2 +/- 2.9 (n = 4) ng/10(6) cells, respectively. The amounts of ANG I and II detected in the media conditioned by a 48-h exposure to cardiomyocytes were 5.2 +/- 1.2 (n = 3) and 2.1 +/- 1.2 (n = 3) ng/10(6) cells, respectively. The amounts of ANG I and II detected in fibroblast cultures (5.38 x 10(6) cells/dish) were 34.8 +/- 4.9 (n = 4) and 8.0 +/- 3.5 (n = 4) ng/10(6) cells, respectively. The amounts of ANG I and II obtained from media conditioned by a 48-h exposure to fibroblasts were 4.7 +/- 0.6 (n = 4) and 3.3 +/- 2.1 (n = 4) ng/10(6) cells, respectively. The identity of the radioimmunoassayable materials as ANG I and II peptides was confirmed in cardiomyocytes using an in vitro bioassay based on displacement of 125I-ANG II from receptor binding sites in cardiac membranes prepared from neonatal pig heart. Identification of ANG I and II and ACE in vitro in cultures of cardiac myocytes and fibroblasts supports the hypothesis that there is an intracardiac renin-angiotensin system that produces these peptides.