Regulation of the biosynthesis and processing of chromogranins in organotypic slices: influence of depolarization, forskolin and differentiating factors.

Slices from rat hippocampus in organotypic culture were used to study the biosynthesis regulation of chromogranins A and B and secretogranin II. Additionally, we investigated the proteolytic conversion of secretogranin II and the levels of prohormone convertases putatively involved. Forskolin treatment and depolarization with potassium plus BayK 8644 led to significant increases in secretogranin II mRNA in the principal cells of the hippocampus. Enhanced expression of secretogranin II was also reflected by a rise in peptide levels. Despite this induction of biosynthesis the extensive processing to secretoneurin normally observed in brain was maintained. Both forskolin and depolarization upregulated the prohormone convertase (PC)1, but not PC2, indicating that PC1 levels are critical for secretoneurin production under stimulating conditions. Results obtained for chromogranins A and B were less consistent. For chromogranin A mRNA, changes were restricted to granule cells; for chromogranin B, a response in granule cells was observed to depolarization but not to forskolin, and effects in pyramidal neurons were weak. Accordingly, we were unable to detect alterations in chromogranin A and B protein levels. Furthermore, we tested several neurotrophic growth factors and found that only basic fibroblast growth factor raised secretogranin II expression without affecting chromogranins A and B. The hippocampal slice preparation allowed well controlled treatment with identification of neuronal subpopulations and yielded data largely matching experiments in vivo and in cell culture. The pronounced regulation of secretogranin II and its effective processing underlines the importance of the resulting peptide secretoneurin as an active neuropeptide in the nervous system.

The new chromogranin-like protein NESP55 is preferentially localized in adrenaline-synthesizing cells of the bovine and rat adrenal medulla.

The protein NESP55, a new member of the chromogranin family, is present in large dense-core secretory granules of neuroendocrine tissues. We investigated its cellular distribution in adrenal medulla with immunohistochemistry and in situ hybridization. A preferential co-localization of NESP55 with phenylethanolamine-N-methyltransferase in the adrenergic cell population was found by immunolabelling of consecutive sections. Noradrenergic cells also contained small amounts of NESP55, but the levels as measured by radioimmunoassay were five times lower. The distribution of NESP55 mRNA was similar to preproenkephalin mRNA which previously was shown to be confined to adrenaline-producing cells of the adrenal medulla. The present study indicates that stimulation of adrenergic cells will release significantly higher amounts of NESP55. The functional implications of this preferential secretion, however, have yet to be discovered.

Relationship of plasma cholesteryl ester transfer protein to HDL cholesterol. Studies in normotriglyceridemia and moderate hypertriglyceridemia.

To evaluate the independent effect of cholesteryl ester transfer protein (CETP) on HDL concentrations in humans, we measured lipids, lipoproteins, postprandial lipemia after an oral fat load, CETP mass, and the activities of CETP, lipoprotein lipase (LPL), and hepatic lipase in 16 healthy, normotriglyceridemic men and in 23 men with moderate, primary hypertriglyceridemia on an American Heart Association Step I diet. Fasting triglycerides and postprandial lipemia were increased and HDL cholesterol (HDL-C) was decreased in hypertriglyceridemic men compared with control subjects (P < .001). In the normotriglyceridemic group, CETP mass (P < .001) and activity (P < .005) were directly related to LPL activity After statistical adjustment for this close association, no significant relationship of CETP to HDL-C independent of LPL activity could be demonstrated in the normotriglyceridemic subjects. In contrast, CETP was unrelated to LPL activity in the hypertriglyceridemic subjects, but CETP concentrations showed a close inverse relationship to HDL-C (r = -.504, P = .014). Structural equation modeling of the association structures between HDL and fasting and postprandial triglycerides, endothelial lipases, and CETP in both groups indicated that the overall regression models for the two groups differed (P < .05). Specifically, the associations between CETP mass and activity and HDL-C differed between both groups (both P < .01). We conclude that high-normal CETP levels lower HDL-C in nonsmoking, nonobese men with moderate, primary hypertriglyceridemia on a hypolipidemic diet, but not in healthy, normotriglyceridemic men on an unrestricted diet. Thus, variation in CETP plasma concentrations may contribute to the high-triglyceride, low-HDL phenotype.

Effects of pancreas transplantation on distribution and composition of plasma lipoproteins.

In type I (insulin-dependent) diabetic patients, peripheral hyperinsulinemia due to subcutaneous insulin treatment is associated with increased high-density lipoprotein (HDL) cholesterol, and also with an altered surface composition of HDL. Pancreas grafts also release insulin into the systemic rather than into the portal venous system, giving rise to pronounced peripheral hyperinsulinemia. We hypothesized that if peripheral hyperinsulinemia is responsible for high HDL cholesterol and/or altered surface composition of HDL in diabetic subjects, similar changes in the lipid profile should be present in pancreas-kidney transplant recipients (PKT-R). Using zonal ultracentrifugation, we isolated HDL2, HDL3, very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), and low-density lipoprotein (LDL) from fasting plasma of 14 type I diabetic PKT-R, eight nondiabetic kidney transplant recipients (KT-R), and 14 healthy control subjects and determined the level and composition of the above lipoproteins. HDL2 cholesterol was increased in PKT-R as compared with KT-R and healthy controls (both P < .05), whereas HDL3 cholesterol was unchanged. However, an altered lipoprotein surface composition was evident in PKT-R: HDL2, HDL3, and LDL were enriched in unesterified cholesterol ([UC] PKT-R v KT-R, P=.13, P < .005, and P < .05, respectively; PKT-R v controls, all P < .005); HDL2 was enriched in phospholipids; and LDL was depleted of phospholipid. KT-R, in contrast, showed no changes in lipoprotein surface composition but a substantial triglyceride enrichment of HDL2 as compared with PKT-R and healthy controls (both P < .05). LDL size as determined by gradient gel electrophoresis was increased in PKT-R compared with controls (P < .005). The plasma concentration of cholesteryl ester (CE) transfer protein (CETP), involved also in phospholipid transfer, was increased in both transplant groups compared with healthy controls (both P < .05). Insulin concentrations in fasting plasma were directly related to CETP levels and to the weight-percentage of UC in HDL3, and inversely to the weight-percentage of phospholipids in LDL (all P < .05). We explain the increase in HDL2 cholesterol and LDL size in PKT-R by their high lipoprotein lipase (LPL) activity conferring an excellent capacity to clear chylomicron triglycerides. Effective handling of postprandial triglycerides, high HDL2 cholesterol, and predominance of LDL pattern A, respectively, are established indicators of a low risk of atherosclerosis. However, it is presently unclear what effects the compositional changes on the surface of HDL and LDL may have on cardiovascular risk in clinically stable PKT-R.

Relationship of high-density lipoprotein subfractions and cholesteryl ester transfer protein in plasma to carotid artery wall thickness.

High plasma concentrations of high-density lipoprotein (HDL) cholesterol are a powerful indicator of low vascular risk. By decreasing HDL cholesterol, cholesteryl ester transfer protein (CETP) could perhaps constitute an atherogenic protein. We measured HDL cholesterol and HDL subfractions and quantified CETP mass in fasting plasma in 21 asymptomatic probands, and related these variables to the mean intima media thickness of the extracranial carotid arteries. HDL2 cholesterol, the less dense HDL subfraction, was inversely related to carotid wall thickness (r = -0.378; P < 0.05), and CETP was directly related to carotid wall thickness (r = 0.436; P < 0.05). In plasma CETP is associated mostly with the HDL3 subfraction. We therefore calculated from our measurements the relative CETP content of HDL3, i.e., CETP/HDL3 cholesterol. This ratio was correlated with carotid wall thickness stronger than any other variable measured (r = 0.718, P < 0.001). We conclude that variation in HDL subfractions and CETP may be more closely associated with carotid intima media thickness than the accepted strong risk factor of HDL cholesterol.