Carlos M. Isales

Immunocytochemical expression and localization of protein kinase C in bovine aortic endothelial cells

Total PKC activity in BAEC incubated for 24 hrs in either 10% serum (FBS) or serum-deprived media (SDM) was similar. However, most of the activity (69%) in the FBS group was detected in the particulate fraction, while it was mainly in the cytosolic fraction (66%) in the SDM group. By confocal microscopy, there was diffuse cytoplasmic localization of the antibodies to the alpha and beta PKC isoforms. gamma PKC was not detected. Treatment of FBS or SDM cells with a phorbol ester resulted in an increase in PKC activity with translocation to the particulate fraction. PKC alpha immunofluorescence redistributed to the perinuclear region whereas PKC beta staining remained mostly cytosolic. Calphostin C, a PKC inhibitor, prevented the phorbol ester-induced increase in PKC activity and translocation.

Cycling of Ca2+ across the plasma membrane as a mechanism for generating a Ca2+ signal for cell activation.

When studies of Ca2+ messenger function were initially carried out in nerve and muscle, it seemed that Ca2+ played its messenger role in a very simple manner: increases and decreases in intracellular Ca2+ concentration led directly to increases and decreases in cellular activity. That is, there was a direct correspondence between changes in CaZ+ concentration and changes in cellular response. However, as studies of Ca2+ messenger function expanded to include a variety of other tissues, it became evident that the Ca2+ messenger system is considerably more complex than originally envisioned. In the present discussion, we will present a brief account of the classic view of Ca messenger function, and then consider the intimate relationship between the Ca and cAMP messenger systems2 This will be followed by a consideration of Ca2+ as a cellular toxin and the important constraint that the threat of Ca2+ intoxication imposes on the use of Ca2+ as intracellular messenger. Finally, the complexity of Ca2+ messenger function will be emphasized by discussing two systems: aldosterone secretion from adrenal glomerulosa cells and insulin secretion from beta cells.3 In the course of this discussion, a particular thesis will be developed: during sustained cellular responses, it is a cycling of Ca2 across the plasma membrane that is the mechanism by which a specific Ca2+ messenger, an increase in the Ca2 concentration in a subdomain of the plasma membrane, [Ca2+],,, is generated. The way in which this Ca2+ messenger leads to a specific cellular response is determined by the nature and number of the Ca2+-sensitive transducers associated with the plasma membrane. In many cases, the cAMP messenger system must be activated appropriately in order for the Ca2+ messages to be effective.

Platelet Cytosolic Calcium, Peripheral Hemodynamics, and Vasodilatory Peptides in Liver Cirrhosis

BACKGROUNDnPlatelet cytosolic calcium concentration ([Ca2+]i) has been proposed to reflect changes in vascular smooth muscle cells. This study investigated if the platelet [Ca2+]i is altered in cirrhotic patients and determined its relationship with peripheral hemodynamics and peptide levels.nnnMETHODSnFourteen patients with cirrhosis and 11 healthy, age- and sex-matched controls had blood samples taken for determining platelet [Ca2+]i and glucagon, substance P (SP), and vasoactive intestinal peptide (VIP) values. Mean arterial pressure (MAP) and forearm blood flow (FBF) were measured on the same day of blood sampling. Forearm vascular resistance (FVR) was calculated.nnnRESULTSnPatients with cirrhosis had lower platelet [Ca2+]i (50.1 +/- 2 vs. 73.0 +/- 5 nmol/L; P < 0.001) than normal controls. Glucagon levels were significantly higher in patients with cirrhosis, but there was no difference in SP or VIP levels in both groups. MAP (80.5 +/- 3 vs. 94.7 +/- 3; P < 0.005) and FVR (20.1 +/- 1 vs. 36.3 +/- 2; P < 0.001) were significantly lower in patients with cirrhosis. A significant correlation was observed between platelet [Ca2+]i and MAP in patients with cirrhosis (r = 0.81; P < 0.001), between platelet [Ca2+]i and FVR (r = 0.87; P < 0.001), and between platelet [Ca2+]i and diastolic blood pressure (r = 0.78; P < 0.001). No correlation was found between platelet [Ca2+]i and peptide levels.nnnCONCLUSIONSnPatients with cirrhosis have a significant reduction in the platelet [Ca2+]i. This finding correlates well with peripheral hemodynamics. Platelets may be a useful tool to study the etiologic mechanisms leading to the vasodilation of chronic liver disease.

Signal transduction mechanisms involved in carbachol-induced aldosterone secretion from bovine adrenal glomerulosa cells.

In cultured bovine adrenal glomerulosa cells, diacylglycerol content remains elevated for up to 75 min following the removal of angiotensin II. This maintained increase could provide a mechanism by which angiotensin II pretreatment may prime cells to secrete aldosterone in response to the calcium channel agonist Bay K 8644. In the present study we find that carbachol failed both to produce this persistent diacylglycerol elevation and to exert a priming effect. In addition, because carbachol was also a less potent activator of phospholipase D than angiotensin II, our results implicate phospholipase D in the maintained increase in diacylglycerol content observed following stimulation with and removal of angiotensin II. Carbachol also elicited changes in the radiolabeled levels of both myristate- and arachidonate-containing diacylglycerol. However, the rapid decline in diacylglycerol content following carbachol removal resembled the rapid fall in arachidonate-diacylglycerol; we therefore proposed that the diacylglycerol species generated with carbachol stimulation contains predominantly arachidonic acid. In summary, our results suggest that prolonged elevations in diacylglycerol content following removal of hormones such as angiotensin II, as well as the identity of the diacylglycerol species itself, may be important in the regulation of cellular responses.

The Cellular Basis for Short-Term Memory in Endocrine Systems

A common property of many endocrine systems is an anamnestic response. For instance, when pancreatic islets are reexposed to a standard glucose stimulus after a prior period of exposure to the same glucose challenge, their insulin secretory response is greater to the second than to the first stimulus (Gold et al., 1982; Grill et al., 1978, 1979; Grill and Rundfeldt, 1979; Grill, 1981; Grodsky et al., 1969). A similar pattern is seen on repeated exposure of adrenal glomerulosa cells to the peptide hormone angiotensin II. Recent work in both of these systems indicates that protein kinase C plays a central role in mediating the sustained phase of each of these endocrine responses (Kojima et al., 1984; Tanigawa et al., 1982; Zawalich et al., 1983, 1984). Work in our laboratories over the past 18 months has focused on the possible role of protein kinase C in this type of short-term cellular memory in bovine adrenal glomerulosa cells and isolated rat pancreatic islets.