Direct action of endotoxin on cardiac muscle.

While the cardiovascular effects of endotoxin include myocardial depression, presumably due to circulating myocardial depressant substances, endotoxin itself is supposed to have no direct effect on the heart. In these experiments, we compared the direct effects of endotoxin on the contractile response of feline papillary muscle to calcium with that seen after administration to the intact animal. The contractile state of the muscle was assessed from developed tension as extracellular calcium concentrations were varied from .5 to 8.0 mM. When endotoxin was administered to the intact animal as well as given to the muscle directly in the bathing solution, endotoxin- exposed muscles showed smaller increases in contractile tension with increasing calcium concentrations than control muscles after 70-85 min. NaOH-inactivated endotoxin produced results similar to the control experiments. Previous experimenters have reported myocardial depression only after endotoxin had circulated in the intact animal for 1-2h; these experiments show a direct depressant effect of endotoxin on cardiac muscle without the release of a substance elsewhere in the body.

Effects of pericardial pressure on systemic and coronary hemodynamics in dogs.

The effects of pericardial tamponade on coronary capacitance and coronary systemic hemodynamics were calculated in two groups of animals subjected to increases in pericardial pressure (PCP) up to approximately 20 mmHg. In one group (A), flow in the left circumflex artery was measured in the intact animal under conditions of increased PCP. In the second group (B), coronary artery perfusion pressure was maintained constant with a pump while PCP was increased. In group A increased PCP was accompanied by a decrease in arterial pressure. This resulted in a marked decrease in coronary blood flow after vasodilation but without a change in coronary vascular resistance. In group B there was no change in coronary flow or coronary vascular resistance with increased PCP. Microsphere distribution to the left ventricular wall showed less endocardial than epicardial flow but no change in going from low to high PCP. Characteristic impedance was altered in the group B animals after vasodilation at medium and high PCP, indicating a loss of reflection sites and probably increased vessel tethering. The coronary artery in a subgroup of group B animals was also perfused by left ventricular pressure, the time constants for coronary backflow showing an 8-12% decrease in capacitance with low and high PCP; these values represent minimal epicardial capacitances vs. total bed capacitance. A diastolic model for the values for resistance and capacitance in the coronary bed is suggested. As expected, most of the capacitance is in the venous bed and most of the resistance is in the arterial bed.

Functional characterization of the rat GAP-43 promoter.

GAP-43 is a highly conserved neuronal protein whose expression is spatially and temporally regulated. Because this regulation may occur, at least in part, at the level of transcription, we have begun to characterize the regions upstream of the GAP-43 transcription unit which direct its neuronal-specific expression. Functional analyses of GAP-43 promoter-reporter constructs have been performed in stably transfected cell lines, including PC12, C6 and RAT2. These data indicate that as little as 600 bp of GAP-43 5'-flanking DNA sequence directs the expression in a neuronal-specific manner. A shorter construct containing 230 bp of 5'-flanking DNA sequence defines a GAP-43 minimal promoter that is active in both neuronal and glial but not in non-neural cell lines. An upstream region, previously shown by other investigators to have promoter activity, was able to stimulate transcription when linked to the downstream minimal promoter. However, this upstream region was by itself unable to direct transcription of the reporting gene. In addition, we have demonstrated that two polypurine tracts within the 5'-flanking DNA sequence of the GAP-43 gene adopt a non-duplex configuration in plasmids, and, when studied in the context of chromosomal integration, these regions have a stimulatory effect on transcription.

Regulation of insulin secretion from beta-cell lines derived from transgenic mice insulinomas resembles that of normal beta-cells.

Insulin secretory physiology has been characterized in tumor cell lines derived by primary culture of insulinomas that developed in transgenic mice expressing the large T-antigen of SV40 in pancreatic islet beta-cells. Cells in one of these lines, beta TC-3, contain large amounts of insulin (3100 +/- 294 ng/100 micrograms cellular protein). Constitutive release of insulin over 2 h in static incubation was low at 31.9 ng/100 micrograms protein and was increased 2-fold by glucose (16.7 mM) and 8-fold by depolarizing concentrations of potassium (45 mM). Isobutylmethylxanthine (IBMX; 0.5 mM) and forskolin (5 and 50 microM), which elevated cellular levels of cAMP, were ineffective as secretagogues, but dramatically potentiated glucose and potassium effects on insulin release (6.5- and 4-fold, respectively). A variety of other known insulin secretagogues stimulated insulin release in a manner analogous to their effects in normal islets. The sulfonylurea glipizide (1 microM) and the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (1 microM) stimulated insulin release 3.4- and 13.7-fold, respectively. The cholinergic agonist carbachol (2 microM) was ineffective alone, but potentiated glucose-induced insulin release 2.8-fold. Comparable stimulation of insulin release by glucose (16.7 mM) and glucose (16.7 mM) plus IBMX (0.5 mM) was noted with several other beta TC lines, which were derived independently from separate transgenic mice. Glucose- and glucose- plus IBMX (0.5 mM)-induced insulin release occurred progressively from 0.15-16.7 mM, indicating that insulin release from beta TC-3 cells occurred at much lower levels than that from normal islets. However, as in the normal islet, the glucose concentration dependency for insulin release was highly correlated (r = 0.93) with the glucose concentration dependency for glucose utilization (measured by 3H2O formation from [5-3H]glucose). This suggests that glucose induces insulin release from beta TC-3 cells by a mechanism similar to that in the normal islet. The high insulin content, the multifold stimulation of insulin release by a variety of secretagogues, their convenient propagation in culture, and the renewable source of these cell lines make the beta TC cells a convenient model for studies of beta-cell function.