Gail Romanchuk

Pituitary adenylate cyclase-activating peptide stimulates neurite growth in PC12 cells

The ability of PACAP-38 to stimulate morphological development was studied using rat pheochromocytoma PC12 cells. PACAP-38 produced concentration-dependent increases in percentage of cells exhibiting neurite extension. Similar increases were produced by forskolin (28 +/- 2% at 96 h) and 8-bromo cAMP (30 +/- 2%). Vasoactive intestinal peptide and alpha-calcitonin gene-related peptide were without effect. PACAP-38 produced significant increases in PC12 cell cAMP content and inositol phosphate turnover. Intracellular [Ca2+] increased from 169 +/- 14 nM to 560 +/- 58 nM in response to 1 microM PACAP-38. PACAP-stimulated neurite outgrowth was abolished by RpcAMPS, an inhibitor of cAMP-dependent kinases but was unaffected by the protein kinase C antagonist H7.

Stimulation of myenteric plexus neurite outgrowth by insulin and insulin-like growth factors I and II

A defined culture medium containing insulin, insulin-like growth factor I (IGF-I) or insulin-like growth factor II (IGF-II) supported morphological development of myenteric plexus neurons derived from neonatal guinea pigs. Insulin increased neurite outgrowth 3-fold at concentrations as low as 0.2 nM. Similar significant and dose-dependent increases in neurite outgrowth were noted with IGF-I and IGF-II. Stimulation of neurite outgrowth was abolished by exposure to cytosine arabinofuranoside, an agent toxic to non-neuronal cells, implying that trophic effects of insulin or insulin-like growth factors require the presence of non-neuronal elements in culture.

Calcium signaling induced by angiotensin II in the pancreatic acinar cell line AR42J

The purpose of this study was to characterize the nature and mechanisms of angiotensin II-evoked calcium signaling in AR42J cells. Cytosolic calcium concentrations were determined using fura-2-based microfluorimetry. Angiotensin II causes elevations in free cytosolic calcium ([Ca2+]i) in the rat pancreatic acinar cell line AR42J. The mechanisms of angiotensin II-evoked calcium signaling were examined using fura-2-based fluorescent digital microscopy. Angiotensin II caused dose-dependent increments in [Ca2+]i over a concentration range of 0.1-1,000 nM, with an average increment of 243 +/- 16 nM at an angiotensin II concentration of 1,000 nM. Dup753, an AT1-specific antagonist, inhibited angiotensin II-evoked signaling, whereas the AT2 antagonist PD123,319 had no effect. Preincubation with the phospholipase C inhibitor U73122 reduced the response in [Ca2+]i to 25% of that of the control. Thapsigargin abolished angiotensin II-evoked calcium signaling. The inositol 1,4,5-trisphosphate receptor antagonist heparin introduced by radiofrequency electroporation inhibited responses to 46 +/- 6% of controls. Angiotensin II-evoked signals were reduced in magnitude and duration by elimination of Ca2+ from the extracellular buffer. Preincubation with pertussis toxin (100 ng/ml) had no effect. Angiotensin II did not stimulate cyclic AMP or suppress vasoactive intestinal peptide stimulated cyclic AMP production over the concentration range that caused Ca2+ signaling.

Reactivation of methionine synthase from Thermotoga maritima (TM0268) requires the downstream gene product TM0269

The crystal structure of the Thermotoga maritima gene product TM0269, determined as part of genome‐wide structural coverage of T. maritima by the Joint Center for Structural Genomics, revealed structural homology with the fourth module of the cobalamin‐dependent methionine synthase (MetH) from Escherichia coli, despite the lack of significant sequence homology. The gene specifying TM0269 lies in close proximity to another gene, TM0268, which shows sequence homology with the first three modules of E. coli MetH. The fourth module of E. coli MetH is required for reductive remethylation of the cob(II)alamin form of the cofactor and binds the methyl donor for this reactivation, S‐adenosylmethionine (AdoMet). Measurements of the rates of methionine formation in the presence and absence of TM0269 and AdoMet demonstrate that both TM0269 and AdoMet are required for reactivation of the inactive cob(II)alamin form of TM0268. These activity measurements confirm the structure‐based assignment of the function of the TM0269 gene product. In the presence of TM0269, AdoMet, and reductants, the measured activity of T. maritima MetH is maximal near 80°C, where the specific activity of the purified protein is ∼15% of that of E. coli methionine synthase (MetH) at 37°C. Comparisons of the structures and sequences of TM0269 and the reactivation domain of E. coli MetH suggest that AdoMet may be bound somewhat differently by the homologous proteins. However, the conformation of a hairpin that is critical for cobalamin binding in E. coli MetH, which constitutes an essential structural element, is retained in the T. maritima reactivation protein despite striking divergence of the sequences.

Dendritic outgrowth of myenteric plexus neurons in primary culture

Myenteric plexus neurons derived from neonatal guinea pigs, when exposed to serum, demonstrated a characteristic pattern of growth, including a proliferating outgrowth zone of glial cells, peripheral extension of dendritic processes, and progressive dendritic growth. Serum effects upon dendritic growth, measured morphometrically, was strongly dose- and temporally dependent. Dendritic density was increased 10-fold (120 hr) by the addition of 6% serum, while mean dendritic length was increased 3-fold. Development of cholinergic function was reflected by release of [3H]ACh in response to cholecystokinin octapeptide, vasoactive intestinal peptide, substance P, and calcitonin gene-related peptide (10(-10) and 10(-8) M).

Small Residue Substitutions at Cardiac Troponin I Ser43/Ser45 Produce Distinct Functional Responses

Our current studies examine the modulatory role of the protein kinase C (PKC)-targeted Ser 43/45 cluster in cardiac troponin I (cTnI). Previously, gene transfer and sarcomeric replacement of endogenous cTnI with phospho-mimetic Asp substitutions at Ser43/Ser45 significantly reduced the peak amplitude and transiently decreased the rate of shortening. Myocytes expressing cTnISer43/45Asp developed an accelerated time to 75% re-lengthening and Ca2+ decay rate. In the current study, we tested whether cTnISer43/45 substitution with Ala would prevent the peak amplitude and shortening rate changes observed with cTnISer43/45Asp. Western blot and immunohistochemistry confirmed that cTnISer43/45Ala gene transfer in adult rat myocytes produced a time-dependent replacement of endogenous cTnI and sarcomeric expression of this cTnI substitution. Unexpectedly, the amplitude and rate of peak shortening in myocytes expressing cTnISer43/45Ala fell between values observed in myocytes expressing endogenous cTnI and cTnISer43/45Asp. These results indicate that additional amino acid properties may be functionally important for these cTnI residues. Next, a cTnISer43/45Asn construct was utilized to test whether polar residues such as Asn serve as a functionally conservative substitution for Ser at these positions. The temporal increase in endogenous cTnI replacement with cTnISer43/45Asn was comparable to cTnISer43/45Ala based on Western analysis and immunohistochemical labeling of myocytes. In ongoing studies, peak shortening amplitude and rate are comparable to controls in myocytes expressing cTnISer43/45Asn, unlike cTnISer43/45Ala. At present, these recent results support the idea that properties other than residue size at Ser43/45 may be important for mimicking the basal shortening phenotype.

Cholinergic Intrapancreatic Neurons Induce Ca2+ Signaling and Early-Response Gene Expression in Pancreatic Acinar Cells

Pancreatic exocrine function has been demonstrated to be under neuronal regulation. The pathways responsible forthis effect, and the long-term consequences of such interactions, are incompletely described. The effects of neuronal depolarization on pancreatic acinar cells were studied to determine whether calcium signaling and c-fos expression were activated. In pancreatic lobules, which contain both neurons and acinar cells, agonists that selectively stimulated neurons increased intracellular calcium in acinar cells. Depolarization also led to the expression of c-fos protein in 24% ±4% of the acinar cells. In AR42J pancreatic acinar cells, cholinergic stimulation demonstrated an average increase of 398 ±19 nmol/L in intracellular calcium levels, and induced c-fos expression that was time and dose dependent. The data indicate that intrapancreatic neurons induce Ca2+ signaling and early-response gene expression in pancreatic acinar cells.