Robert W. Wrenn

Phorbol ester induces intracellular translocation of phospholipid/Ca2+-dependent protein kinase and stimulates amylase secretion in isolated pancreatic acini.

Treatment of intact rat pancreactic acini with phorbol ester (12‐O‐tetradecanoyl‐phorbol‐13‐acetate, TPA) resulted in a time‐ and concentration‐dependent translocation of phospholipid/Ca2+ ‐dependent protein kinase (PL/Ca‐PK) from the soluble fraction. Redistribution of PL/Ca‐PK was concurrent with stimulation of amylase secretion by TPA‐treated acini. Polymyxin B, a potent and selective inhibitor of PL/Ca‐PK completely inhibited TPA‐induced amylase secretion. These findings are consistent with a role for PL/Ca‐PK in the regulation of pancreatic exocrine secretion.

Direct effect of the neurotoxicant acrylamide on kinesin-based microtubule motility

Acrylamide (ACR) is an environmental toxicant and prototypic tool for studying mechanisms of peripheral neuropathies. Reductions in fast anterograde axonal transport (faAXT) are thought to be a critical step leading to axonal degeneration. Kinesin and microtubules (MT) were evaluated as molecular sites of action using an in vitro MT motility assay. The number of locomoting MT which lifted from a bed of kinesin (MT detachments or MTD), increased from 7% in controls to 80, 89, and 100% following preincubation of kinesin (37°C, 20 min) with 0.1, 0.5, or 1.0 mM ACR, respectively; rates were variably reduced by as much as 20%. Similar alterations were observed with N‐ethylmaleimide. A non‐neurotoxic analogue, propionamide (1mM), had no effect on either parameter. Preincubation of taxol‐stabilized MT with ACR produced a dose‐dependent increase in MTD but no changes in rate. We conclude that kinesin and MT are covalently modified by ACR resulting in reduced affinity for each other. The greater sensitivity of kinesin indicates that a primary cause of transient, ACR‐induced reductions in faAXT is covalent modification of kinesin. Such reductions in faAXT may be sufficient to produce axonal degeneration. Further, ACR may prove useful as a pharmacological tool to decipher the complex mechanics of kinesin‐MT interactions.

Dual calcium-dependent protein phosphorylation systems in pancreas and their differential regulation by polymyxin B☆

Both phospholipid/calcium (PL/Ca2+) activated and calmodulin/Ca2+ (CaM/Ca2+)activated protein kinase systems were found in rat pancreatic extracts treated with Sephadex G-25. At least four substrate proteins for PL/Ca2+-activated kinase and one for a CaM/Ca2+-activated kinase were noted. Polymyxin B, an amphipathic antibiotic, was over 100-fold more potent as an inhibitor of PL/Ca2+-dependent protein phosphorylation than of the CaM/Ca2+-dependent system (Ki = app. 7 microM v. 950 microM). Fluphenazine inhibited both PL/Ca2+- and CaM/Ca2+-dependent protein kinases with equal potency, as did dibucaine. Inhibition by polymyxin B of PL/Ca2+-dependent phosphorylation could be overcome by increased amounts of phosphatidylserine. Low concentrations (10(-5)M) of polymyxin B completely inhibited carbachol-stimulated amylase release from intact pancreatic acini. These results indicate that polymyxin B may be useful in delineating the relative roles of PL/Ca2+-dependent and CaM/Ca2+-dependent protein phosphorylation in biological systems and suggest a potential role for the PL/Ca2+-activated kinase in regulation of pancreatic exocrine function.

Additive effect of mifepristone and tamoxifen on apoptotic pathways in MCF-7 human breast cancer cells.

MCF-7 cells growing in culture were used to study the mechanism of the antiproliferative activity of the antiprogestin mifepristone, as compared with the antiestrogen 4-hydroxytamoxifen or the combination of both. These steroid antagonists induced a significant time- and dose-dependent cell growth inhibition (cytotoxicity). This inhibition of cell survival was associated with a significant increase in DNA fragmentation (apoptosis), downregulation of bcl2, and induction of TGFβ1 protein. Abrogation of the mifepristone- and/or 4-hydroxytamoxifen-induced cytotoxicity by TGFβ1 neutralizing antibody confirms the correlation between induction of active TGFβ1 and subsequent cell death. The effect of a combination of mifepristone and 4-hydroxytamoxifen on cell growth inhibition, on the increase in DNA fragmentation, bcl2 downregulation, and induction of TGFβ1 protein was additive and significantly different (P < 0.05) from the effect of monotherapy. A translocation of protein kinase C (PKC) activity from the soluble to the particulate and/or nuclear fraction appeared to be also additive in cells treated with a combination of both 4-hydroxytamoxifen and mifepristone. These results suggest that the mechanism of the additive antiproliferative activity of mifepristone and tamoxifen could be explained at least in part by an additive induction of apoptosis in both estrogen and progesterone receptor positive MCF-7 breast cancer cells. A bcl2 downregulation, the PKC transduction pathway, and TGFβ1 expression seem to be involved in this additive mechanism of action. Our data further suggest that a combination of an antiprogestin with tamoxifen may be more effective than tamoxifen monotherapy in the management of human breast cancer.

Phosphorylation of a pancreatic zymogen granule membrane protein by endogenous calcium/phospholipid-dependent protein kinase

The occurrence of phospholipid-sensitive calcium-dependent protein kinase (referred to as C kinase) and its endogenous substrate proteins was examined in a membrane preparation from rat pancreatic zymogen granules. Using exogenous histone H1 as substrate, C kinase activity was found in the membrane fraction. The kinase was solubilized from membranes using Triton X-100 and partially purified using DEAE-cellulose chromatography. An endogenous membrane protein (Mr approximately equal to 18 000) was found to be specifically phosphorylated in the combined presence of Ca2+ and phosphatidylserine. Added diacylglycerol was effective in stimulating phosphorylation of exogenous histone by the partially purified C kinase, but had no effect upon phosphorylation of the endogenous 18 kDa protein by the membrane-associated C kinase. Phosphorylation of the 18 kDa protein was rapid (detectable within 30 s following exposure to Ca2+ and phosphatidylserine), and highly sensitive to Ca2+ (Ka = 4 microM in the presence of phosphatidylserine). These findings suggest a role for this Ca2+-dependent protein phosphorylation system in the regulation of pancreatic exocrine function.

Transforming growth factor-beta: Signal transduction via protein kinase C in cultured embryonic vascular smooth muscle cells

SummaryTransforming growth factor-beta (TGF-β), an ubiquitous regulatory peptide, has diverse effects on the differentiation and behavior of vascular smooth muscle cells (VSMC). However, the molecular mechanism through which TGF-α exerts its effects remains obscure. We investigated the phosphoinositide/protein kinase C [PKC] signaling pathway in the action of TGF-β on cultured embryonic avian VSMC of differing lineage: a) thoracic aorta, derived from the neural crest; and b) abdominal aorta, derived from mesenchyme. The second messenger responsible for activation of PKC is sn-1,2-diacylglycerol [DAG]; TGF-β increased the mass amounts of DAG in the membranes of neural crest-derived VSMC concurrent with translocation of PKC from the soluble to the membrane fraction, but TGF-β had no effect on the DAG or PKC of mesenchyme-derived VSMC. TGF-β potentiated the growth of platelet-derived growth factor (PDGF)-treated, neural crest-derived VSMC; but abolished PDGF-induced growth of mesenchymal cells. It is concluded that molecular and functional responses of VSMC to TGF-β are heterogeneous and are functions of the embryonic lineage of the VSMC.

Linoleic acid is a potent activator of protein kinase C type III- α isoform in pancreatic acinar cells; its role in amylase secretion

Linoleic acid, an unsaturated-long chain fatty acid, was found to maximally activate protein kinase C (PKC) more effectively than arachidonic or linolenic acid, while the saturated fatty acids palmitic or arachidic had no stimulatory effect. Treatment of intact pancreatic acinar cells with linoleic acid resulted in dose-dependent phosphorylation of endogenous substrate proteins for this kinase and simultaneously stimulated amylase secretion in a dose- and time-dependent fashion. During chromatographic separation of pancreas protein kinase C activity, utilizing hydroxylapatite (HTP), Type III-alpha PKC isoform was detected. These data are consistent with a role for PKC in the regulation of pancreatic exocrine secretion.

Identification of a major endogenous substrate for phospholipid/Ca2+-dependent kinase in pancreatic acini as Gc (vitamin D-binding protein)

A major 56 kDa substrate for phospholipid/Ca2+‐dependent kinase (C‐kinase) in pancreatic acinar cells is physicochemically and immunologically indistinguishable from the vitamin D‐binding protein, Gc or group‐specific component. Cellular Gc was also phosphorylated in intact cells following treatment with carbachol as a physiological stimulus. These findings indicate the potential usefulness of Gc as a defined substrate for further studies of the biological role of C‐kinase activity in pancreatic acini and possibly in other cells.

Phospholipid-sensitive calcium-dependent protein kinase and its endogenous substrate proteins in rat pancreatic acinar cells☆

Phospholipid-sensitive Ca2+-dependent protein kinase and its endogenous substrate proteins were examined in acinar cells from rat pancreas. The enzyme was clearly demonstrable by DEAE-cellulose chromatography of acinar cell extract. At least four endogenous substrate proteins (Mr = 38K, 30K, 22K and 15K) for this Ca2+-activated kinase were found in the acinar cell extract. These substrate proteins were maximally phosphorylated in the combined presence of Ca2+ and phosphatidylserine. Calmodulin was partially effective as a cofactor for phosphorylation of the 38K substrate protein, but ineffective for the other three. A slight Ca2+/phospholipid-dependent phosphorylation of 38K and 30K proteins, but not of 22K and 15K proteins was seen in extract of isolated pancreatic islets. The Ka for Ca2+ for phosphorylation of the endogenous acinar cell proteins was decreased more than ten-fold in the combined presence of phosphatidylserine and unsaturated diacylglycerol. The presence of this Ca2+/phospholipid-dependent protein kinase/protein phosphorylation system provides a potential mechanism of action for Ca2+ as a regulator of exocrine pancreatic function.

Inhibition of estrogen stimulated mitogenesis by 3-phenylacetylamino-2,6-piperidinedione and its Para-hydroxy analog

3-Phenylactetylamino-2,6-piperidinedione (A10) inhibited estradiol stimulated cell growth in the MCF-7 (E3) human breast tumor cell line in vivo and in vitro. While high concentrations of A10 were needed to inhibit cell proliferation (IC50 = 3 x 10(-3) M in vitro), the compound demonstrated little toxicity. The effect appeared specific since a hydrolysis product of A10, phenylacetylglutamine, demonstrated no growth inhibitory activity at similar concentrations in MCF-7 (E3) cells in vitro. A computer designed analog, p-hydroxy A10, was more potent than A10 in inhibiting activity in MCF-7 (E3) cells in vitro. The IC50 for p-hydroxy A10 was 7 x 10(-6) M which was comparable to that of the antiestrogen, tamoxifen (IC50 1 x 10(-7) M). All three compounds caused a decline in estrogen receptor levels in a dose-dependent fashion. A10 also inhibited estradiol induction of progesterone receptors. Examination of protein kinase activity following an acute exposure to a 10(-11) M growth stimulatory dose of estradiol revealed a 168% increase in protein kinase activity over that of untreated control cells. A10 in a dose-responsive fashion inhibited the estradiol stimulated increase in protein kinase activity. The protein kinase activity was also inhibited by p-hydroxy A10. These activities of A10 and p-hydroxy A10 coupled with the low toxicity and novelty of the basic A10 structure provide an exciting possibility of developing a new class of clinically useful antineoplastic drugs with minimal side effects.