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After loss of intestinal surface area, the remaining bowel undergoes a morphometric and functional adaptive response. Enterocytic expression of the transcriptional coregulator tetradecanoyl phorbol acetate induced sequence 7 (Tis7) is markedly increased in a murine model of intestinal adaptation. Mice overexpressing Tis7 in intestine have greater triglyceride absorption and weight gain when fed a high-fat diet (42% energy) than their wild-type (WT) littermates fed the same diet. These and other data suggest that Tis7 has a unique role in nutrient absorptive and metabolic adaptation. Herein, male [Tis7.sup.-/-] and WT mice were fed a high-fat diet (42% energy) for 8 wk. Weight was monitored and metabolic analyses and hepatic and intestinal lipid concentrations were compared after 8 wk. Intestinal lipid absorption and metabolism studies and intestinal resection surgeries were performed in separate groups of [Tis7.sup.-/-] and WT mice. At 8 wk, weight gain was less and jejunal mucosal and hepatic triglyceride and cholesterol concentrations were lower in [Tis7.sup.-/-] mice than in the WT controls. Following corn oil gavage, serum cholesterol, triglyceride, and FFA concentrations were lower in the [Tis7.sup.-/-] mice than in the WT mice. Incorporation of oral [sup.3][H] triolein into intestinal mucosal cholesterol ester and FFA was less in [Tis7.sup.-/-] compared with WT mice. Following resection, crypt cell proliferation rates and villus heights were lower in [Tis7.sup.-/-] than in VVT mice, indicating a blunted adaptive response. Our results suggest a novel physiologic function for Tis7 in the gut as a global regulator of lipid absorption and metabolism and epithelial cell proliferation. J. Nutr. 140: 1907-1914, 2010. doi: 10.3945/jn.110.127084.

Ileal apical [Na.sup.+]-dependent bile acid transporter (ASBT) is responsible for reabsorbing the majority of bile acids from the intestinal lumen. Rapid adaptation of ASBT function in response to physiological and pathophysiological stimuli is essential for the maintenance of bile acid homeostasis. However, not much is known about molecular mechanisms responsible for acute posttranscriptional regulation of ileal ASBT. The protein kinase C (PKC)-dependent pathway represents a major cell signaling mechanism influencing intestinal epithelial functions. The present studies were, therefore, undertaken to investigate ASBT regulation in intestinal Caco-2 monolayers by the well-known PKC activator phorbol 12-myristate 13-acetate (PMA). Our results showed that [Na.sup.+]-dependent [[sup.3]H]taurocholic acid uptake in Caco-2 cells was significantly inhibited in response to 2 h incubation with 100 nM PMA compared with incubation with 4[alpha]-PMA (inactive form). The inhibitory effect of PMA was blocked in the presence of 5 [micro]M bisindolylmaleimide I (PKC inhibitor) but not 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM ([Ca.sup.2+] chelator) or LY-294002 (phosphatidylinositol 3-kinase inhibitor). PMA inhibition of ASBT function was also abrogated in the presence of myristoylated PKC[zeta] pseudosubstrate peptide, indicating involvement of the atypical PKC[zeta] isoform. The inhibition by PMA was associated with a significant decrease in the maximal velocity of the transporter and a reduction in ASBT plasma membrane content, suggesting a modulation by vesicular recycling. Our novel findings demonstrate a posttranscriptional modulation of ileal ASBT function and membrane expression by phorbol ester via a PKC[zeta]-dependent pathway. apical sodium-dependent bile acid transporter; SLC10A2; diacylglycerol; atypical protein kinase C[zeta]

Results show that while 1,2-diacyl-sn-glycerol is capable of inducing protein kinase C, 1-O-hexadecyl-2-acetyl-sn-glycerol (HAG), as a 'nonactivating' enzyme, modulates protein kinase C by interacting with the low-affinity phorbol ester binding site.

A testicular angiotensin-converting enzyme (ACE) was mutated by replacing the stalk with an epidermal growth factor (EGF)-domain (ACE-JMEGF) for the purpose of proving that the membrane-protein-solubilizing protease cleavage necessitated a juxtamembrane stalk. The ACE-JMEGF was poorly released on the cell surface and that the release was caused by phorbol ester and regulated by the tumor necrosis factor-alpha protease inhibitor. Poor release of the enzyme was due to the residual disulfide tethering of the ectodomain to the membrane-enclosed C-terminal stump.

A study was conducted to exploit the robust positive inotropic response produced by photogeneration of diacylglycerol to characterize protein kinase C -dependent mechanisms with good signal-to-noise ratio in living cardiomyocytes. It was found that the vast majority of the positive inotropic responses to diacylglycerol is due to enhancement of systolic Ca2+ as a result of stimulation of the process of excitation-contraction coupling.

A study was conducted to examine how protein kinase C with phorbol 12-myristate 13-acetate (PMA) influence the time-dependent changes of apical membrane epithelial Na+ channels. Isolated epithelial of frog skin and connective tissue of the corium were determined from the abdominal skins of R. pipiens pipiens. Results indicated that the effects of time of exposure on transport and time course were not reversible by omission of PMA from the bathing solution.

The effects of phorbol esters, epidermal growth factor (EGF) and angiotensin (ANG) II on the translocation of the alpha, delta and epsilon isozymes of protein kinase C in rat liver epithelial cells were investigated through Western blot analysis following subcellular fractionation of treated tissues. Phorbol esters induced the highest degree of membrane translocation of the different isozymes. Although the magnitude of translocation induced by both EGF and ANG II is low, the significant translocation of the delta and epsilon isozymes is enough to explain spatially defined effects.

Tumor necrosis factor-alpha (TNF-alpha) and phorbol ester inhibits the synthesis of surfactant proteins A and B. Inhibition studies were undertaken to determine the role of the transcription factor nuclear factor-kappaB (NF-kappaB) on the downregulation of surfactant protein synthesis. Results indicate that NF-kappaB does not mediate the downregulation of surfactant protein synthesis by TNF-alpha and phorbol ester.

The function of protein kinase C (PKC) in the regulation of organic anion (fluorescein, FL) transport in killifish renal proximal tubules was examined through fluorescence microscopy and digital image analysis. Phorbol ester decreased cellular and luminal accumulation of FL and protein kinase inhibitors increased cellular and luminal accumulation. The phorbol ester induced PKC translocation from cytoplasm to the plasma membrane. Results showed that renal organic anion transport is negatively correlated with PKC activity and that PKC directly or indirectly regulates the basolateral step in the transport.

The mechanisms by which phorbol 12-myristate 13-acetate (PMA) could inhibit sickle red blood (SS) cell dehydration were characterized. The study revealed that the expression of protein kinase C (PKC) alpha and PKC-zeta are at higher levels in SS cells. Meanwhile, PMA was found to stimulate calcium efflux in deoxygenated SS cells by direct activation of the calcium pump. PMA was also found to inhibit potassium efflux in SS cells which may due to decreased deoxygenation-induced rise in cytosolic calcium.

The role of protein kinase C (PKC) in the regulation of surfactant secretion was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblot techniques in pathogen-free male Sprague-Dawley rats. PKC isozymes were identified in rat alveolar type II cells by RT-PCR and immunoblot analysis. Administration of phorbol ester also activated group A and group B isozymes in a dose-dependent manner indicating the role of intracellular Ca2+ and PKC-alpha and -beta isozymes in stimulating surfactant secretion from alveolar type II cells.

The role of P-selectin in mediating the mechanisms of small cell lung carcinoma (SCLC) cellular adhesion with activated endothelial cells during metastasis was analyzed by utilizing H82 SCLC cell lines. The H82 cells exhibited enhanced free Ca2+-dependent attachment to endothelial cells that are activated by phorbol ester 12-omicron-tetradecanoylphorbol-13-acetate (TPA). However, the adhesion of H82 cells to TPA-activated EC was mediated by P-selectin expression which bind with the cell surface sialic acid residues of SCLC.

Phorbol esters mediate the regulatory effects of protein kinase C on the p-aminohippurate (PAH) transport in opossum kidney (OK) cells by activating protein kinase C. Phorbol 12-myristate 13-acetate (PMA) exhibits an inhibitory effect on the transcellular transport of PAH from the basal to the apical side of cell monolayers, and on the accumulation of PAH in the cells. Diacylglycerols also activate the protein kinase C through an inhibitory process. However, the inactive phorbol ester, 4alpha-phorbol 12,13-didecanoate has little effect on the protein kinase C.

The apoptosis in IL-6-starved T1165 and T1198 plasmacytoma cell lines is suppressed through phorbol ester (PMA) treatment in a protein kinase C (PKC) -mediated process involving alpha/delta isozymes. The PMA-induced PKC activation suppresses apoptosis only when the cells are in a competent state. The cells are competent for apoptosis suppression either immediately after IL-6 withdrawal, or prior to start of apoptosis. Treatment of the cells with PMA or PKC stimulators increases the amount of cells present in the S phase of the growth cycle.

The effects of phorbol esters on L-type calcium channel currents in guinea pig ventricular myocytes were investigated by monitoring the responses of potassium current and chloride current to active phorbol 12-myristate 13-acetate and forskolin. The presence of protein kinase C (PKC) inhibited L-type calcium current three times faster than comonitored stimulation of potassium, L-type calcium and L-type sodium currents. It is suggested that inhibition of calcium current by phorbol esters is mediated by PKC.

The role of phorbol 12-myristate 13-acetate (PMA) in the regulation of the inhibitory effect of phorbol esters on cell growth was investigated using rat vascular smooth muscle (VSM) cells. Exposure to PMA reduced the aggregation of phosphatidylinositol 3-kinase (P13K) to the fibroblast growth factor receptor and tyrosine phosphorylation of the regulatory domain of P13K by 94% and 79%, respectively. P13K catalytic activity also decreased 88%. In contrast, addition of P13K inhibitor wortmannin attenuated cell growth in a dose-dependent manner. The results suggest that the inhibitory effect of phorbol esters in VSM cells occurs upstream of signal transfer protein aggregation.

Phorbol 12-myristate 13-acetate (PMA) increases the mRNA and protein level of constitutively expressed prostaglandin H synthase-1 (PGHS-1) in cultured human umbilical vein endothelial cells. The increase of PGHS-1 depends on time and concentration of PMA present in the cultured cells. The increase in PGHS-1 correlates to an increase in the 5'-promoter activity of PGHS-1. PMA probably increases PGHS-1 by activating protein kinase C, as the increase is inhibited by suppressors of protein synthesis.

Isolated rat cardiac myocytes were treated with lipopolysaccharide (LPS) and phorbol ester to determine if protein kinase C is involved in LPS-induced and nitric oxide synthase (NOS)-dependent impairment of contractility in cardiac myocytes. The results show that phorbol ester and LPS reduce cardiac myocyte contractility by activating PKC, which in turn stimulates NOS expression.

The interaction of phosphatidylinositol 3-kinase (PI3) with CD28, the surface receptor of T-lymphocytes, and the immunoprecipitation of p85 PI3 kinase by anti-phophotyrosine antibodies is inhibited when Jurkat T cells are treated with phorbol 12-myristate 13-acetate (PMA). PMA also inhibits the activation of PI3 kinase by the anti-CD28 monoclonal antibody 9.3. However the formation of interleukin 2 after the activation of Jurkat T cells by the anti-CD28 monoclonal antibodies and PMA is unaffected by the inhibitor of PI3 kinase.

The binding domain for phorbol 13-acetate on the cys2 domain of protein kinase C-delta (PKC-delta) is formed by pulling apart the two beta-sheets and five hydrogen bonds are formed between the phorbol and PKC-delta. The binding of phorbol to the protein forms a hydrophobic surface which extends over 1/3 of the domain and the formation of the hydrogen bonds and the free energy released helps in the insertion of the protein into the membrane. The insertion of the unliganded protein into the membrane is unfavorable because hydrogen bonds at the membrane-protein interface are not formed.

A study of the adenosine receptor subtype that mediates protein kinase C (PKC) regulation in porcine coronary artery by comparing the contractile responses to endothelin-1 with phorbol 12, 13-dibutyrate (PDBu) with or without adenosine receptor antagonists and agonists reveals that A1-adenosine receptors in the coronary artery promote PKC regulation by adenosine analogues. While incubation with PDBu alone does not generate contractions in response to ET-1, an A1-receptor agonist ENBA lowers the effect of PDBu on ET-1 contractions, and incubation with ENBA alone doubles ET-1 contractility. Measurement of arterial PKC activities reveals that enzyme activity doubles with exposure to ENBA, while it remains unaltered on exposure to an A2-receptor agonist CGS-21680.

A study of the kinetic properties of Na+ H+ exchange in vascular smooth muscle cells (VSMC) of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats pretreated with phorbol 12-myristate 13-acetate (PMA) reveals that PMA reduces Hill coefficient and enhances maximal activity in the SHR compared to WKY rats. PMA does not affect the kinetic variables of Na+ H+ antiport in the VSMC of SHR indicating a significant level of antiporter prestimulation.

Immunoblotting and electrophoresis experimental analyses of adenylyl cyclase 2 (AC2) expression in Sf9 cells reveal that protein kinase C stimulation leads to activation and phosphorylation of AC2. Physiologically detectable Mg2+ levels and phorbol 12-myristate 13-acetate treatment enhance the basal activity of AC2 and 32P labeling induces a three-fold rise in the insertion of 32P into epitope-tagged AC2. Covalent changes in the cell surface effector enzyme helps develop an independent signal transmission mode through the G protein pathways.

PCR cloning helps identity a serine/threonine protein binding tumor-promoting phorbol ester and a diacylglycerol, protein kinase D (PKD). Sequential analysis shows that PKD has membrane localization signals and a cysteine-rich repeat sequence homologous to that in the regulatory domain of protein kinase C (PKC). Bacterially expressed catalytic domain of PKD exhibits increased phorbol ester binding activity. The sequence of the catalytic domain of PKD is similar to that of serine protein kinase and shows no similarity to those of PKCs.

Phorbol 12-myristate 13-acetate (PMA)-induced enhancement of intracellular calcium in endothelial cells occurs due to influx of Ca2+ via voltage-influenced L-type Ca2+ channels. The calcium response is restricted by L-channel selective antagonists, increased by depolarizing conditions and influenced by extracellular Ca2+. Protein kinase C-controlled phosphorylation may play a direct role in enhanced Ca2+ influx in endothelial cells.

Study of the influence of the phorbol ester, phorbol myristate acetate (PMA), on the ATP-sensitive K+ channel activity reveals that PMA modulates protein kinase in insulin secreting cells, resulting in mixed effects on K+ATP channels. The initial closure effect is followed by activation of K+ channels in the presence of intracellular ATP. Two PKC isoforms are associated with the K+ATP channel functions that produce biphasic effects on the regulation of channel activity.

Prostaglandin levels measured using enzyme immunoassay in piglets reveal that cortical astroglia release prostaglandins into extracellular-cerebro spinal fluids, where it influences the cerebrovascular tone. Phorbol 12, 13 - dibutyrate (PDB) liberates arachidonic acid to increase the protein kinase C-induced activation of phospholipase A2, which enhances the production of prostaglandin.

Phorbol 12-myristate 13-acetate (PMA) increases phosphorylation, function and expression of protein-tyrosine-phosphatase (PTP) 1C in HL-60 promyelocytic human leukemia cells. PTP1C contributes to 70% of the total phosphate activity and localizes on the plasma membrane. The plasma membrane's translocation and induction period are associated with the differentiation process.

Analysis of the influence of phorbol 12-myristate 13-acetate (PMA) incited megakaryocytic differentiation on expression of genes in cultured CHRF-288 cells reveals that during megakaryocytic differentiation, CHRF-288 cells modulate the gene expression for oncogenes, receptors and platelet proteins. Gene expression in both megakaryocyte differentiation and megakaryoblast proliferation exhibits a different pattern.

A model of cultured human intestinal epithelial monolayer disassembly by continued exposure to phorbol ester 12-O-tetradecanoylphorbol 13-acetate is described. Protein kinase C (PKC) levels diminish as a result of continued exposure of phorbol ester in many cell types such as T84. T84 monolayers dissociate into two or three rounded cells under PKC down regulated conditions, and these morphological studies are not considered during proliferation. Disassembled monolayers are more permeable than the intact monolayers.

The effect of protein kinase C on secretion of GH3 pituitary cells to Ca2+ was studied. Observations reveal that protein kinase C activation with 100nM 12-O-tetradecanoylphorbol 13-acetate TPA for 40 min blocked the increase in intracellular Ca2+ caused by depolarization. Half-maximal secretion of prolactin was also observed at lower Ca2+ concentrations compared to TPA-treated cells. The study concludes that protein kinase C activation boosts the Ca2+ sensitivity of the secretory response of GH3 cells which results in a twofold increase in secretion rate of intracellular Ca2+.

The effects of phorbol 12-myristate 13-acetate (PMA) with 4beta-phorbol 12,13-dibutyrate (PdBu) in stimulating protein discharge in the lacrimal glands of rats were studied. Results show that PMA is less efficient compared to PdBu. Tests proved that neither sphingosine, chelerythrine, staurosporrine failed to inhibit both PMA and PdBu. On the other hand, trifluoperazine produced complete inhibition of PMA-induced protein discharge. The study questions the identification of protein kinase C as sole effector of PMA-stimulated protein secretion in rat lacrimal glands.

The roles of protein kinase C, dopamine DA-1 receptors and cyclic adenylic acid (cAMP) in vasodilation and blood pressure regulation were investigated. The study protocol involved examination of the interaction between a phorbol ester and DA-1 receptors in rat vascular smooth muscle cells. The results indicate that protein kinase C induces an increase in the expression of DA-1 receptors, thus enhancing receptor-coupled generation of cAMP.

The effect of the phorbol 12-myristate 13-acetate (PMA) on the secretion of cyclic adenylic acid (cAMP) and Cl- was investigated using epithelial preparations of the colonic carcinoma cell line T84. The results showed that there is no Ca-dependent ion channel in the apical membrane, and that only Ca-dependent agonists can activate K-channels in the basolateral membrane. Furthermore, PMA inhibition of cAMP- and Ca-dependent Cl- secretion is due to inhibition of K-channels in the basolateral membrane.

CHO cell lines overexpressing various subspecies of protein kinase C (PKC) were developed and monitored for alterations in the cell cycle upon stimulationwith phorbol 12-miristate 13-acetate. The results showed that overexpression ofPKC-delta subspecies resulted in cell division arrest. The phase of arrest was localized to the final step of cytokinesis, resulting into telophase-like dikaryons. Northern blot analysis showed that the delta subspecies is present in various cell types, and may be a regulatory factor controlling the cell cycle under physiological conditions.

A45 kiloDalton phorbol ester-stimulated protein kinase that phosphorylates and activates the extracellular-signal regulated kinase 1(Erk-1) gene product is purified to near homogeneity. This kinase is called the mitogen-activated protein kinase (MAPK)/Erk kinase (MEK). Similarity to the proteins encoded by the Schizosaccharomyces pombe byr1 and Saccharomyces cerevisiae STE7 genes was evident from the sequence analysis of peptides produced by trypsin digestion of MEK.

Phorbol 12-myristate 13-acetate (PMA) treatment of U-937 human leukemic cells induces differentiation to macrophages accompanied by increased expression of the protooncogenes JUN and JUNB. However, mobility shift comparisons showed that PMA phosphorylates only JUN, although the synthesis of both JUN and JUNB increases in response to PMA. Mutation analysis localized the phosphorylation sites on JUN at Ser-63 and Ser-73. In addition, reporter gene fusions demonstrated a transcriptional activation domain within the N-terminal 84 amino acids of JUN.

The v-jun and c-jun oncogenes produce homologous proteins which are capable of forming heterodimers with Fos protein and activating transcription of AP-1-responsive genes. The effects of phorbol esters on the activities of these oncogenes were examined in human leukemic cells. The results showed that phorbol esters stimulated an increase in phosphorylation of c-Jun, but not v-Jun. Mutation analysis localized the delta domain of c-Jun as critical to phorbol ester-induced N-terminal phosphorylaton.

The role of protein kinase C (PKC) in the cytoskeletal reorganizations underlying cell shape changes and membrane ruffling in activated neutrophils was investigated by studying the effects of phorbol esters on actin assembly and on PKC activity inhuman neutrophils. The results showed that phorbol esters induce actin assembly, a process mediated not by conventional PKC isoforms but by novel isoforms such as nuclear PKC.

We investigated the mechanism of the inhibitory action of phorbol 12,13-dibutyrate (PDBu), one of the typical protein kinase C (PKC) activators, in in vitro smooth muscle strips and in isolated smooth muscle cells of the opossum internal anal sphincter (IAS). The inhibitory action of PDBu on IAS smooth muscle (observed in the presence of guanethidine + atropine) was partly attenuated by tetrodotoxin, suggesting that a part of the inhibitory action of PDBu is via the nonadrenergic, noncholinergic neurons. A major part of the action of PDBu in IAS smooth muscle was, however, via its direct action at the smooth muscle cells, accompanied by a decrease in free intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) and inhibition of PKC translocation. PDBu-induced IAS smooth muscle relaxation was unaffected by agents that block [Ca.sup.2+] mobilization and Na.sup.+]-[K.sup.+]-ATPase. The PDBu-induced fall in basal IAS smooth muscle tone and [[[Ca.sup.2+]].sub.i] resembled that induced by the [Ca.sup.2+] channel blocker nifedipine and were reversed specifically by the [Ca.sup.2+] channel activator BAY K 8644. We speculate that a major component of the relaxant action of PDBu in IAS smooth muscle is caused by the inhibition of [Ca.sup.2+] influx and of PKC translocation to the membrane. The specific role of PKC downregulation and other factors in the phorbol ester-mediated fall in basal IAS smooth muscle tone remain to be determined. smooth muscle tone; smooth muscle cells; phorbol esters; calcium influx; calcium channel activator

Repulsive guidance cues can either collapse the whole growth cone to arrest neurite outgrowth or cause asymmetric collapse leading to growth cone turning. How signals from repulsive cues are translated by growth cones into this morphological change through rearranging the cytoskeleton is unclear. We examined three factors that are able to induce the collapse of extending Helisoma growth cones in conditioned medium, including serotonin, myosin light chain kinase inhibitor, and phorbol ester. To study the cytoskeletal events contributing to collapse, we cultured Helisoma growth cones on polylysine in which lamellipodial collapse was prevented by substrate adhesion. We found that all three factors that induced collapse of extending growth cones also caused actin bundle loss in polylysine-attached growth cones without loss of actin meshwork. In addition, actin bundle loss correlated with specific filamentous actin redistribution away from the leading edge that is characteristic of repulsive factors. Finally, we provide direct evidence using time-lapse studies of extending growth cones that actin bundle loss paralleled collapse. Taken together, these results suggest that actin bundles could be a common cytoskeletal target of various collapsing factors, which may use different signaling pathways that converge to induce growth cone collapse. Key words: growth cone * collapse * actin filaments * actin bundles * axon guidance

Longo, Nicola, Fernando Scaglia, and Yuhuan Wang. Insulin increases the turnover rate of [Na.sup.+]-[K.sup.+]-ATPase in human fibroblasts. Am J Physiol Cell Physiol 280: C912-C919, 2001.--Insulin stimulates [K.sup.+] transport by the [Na.sup.+]-[K.sup.+]-ATPase in human fibroblasts. In other cell systems, this action represents an automatic response to increased intracellular [[Na.sup.+]] or results from translocation of transporters from an intracellular site to the plasma membrane. Here we evaluate whether these mechanisms are operative in human fibroblasts. Human fibroblasts expressed the [[Alpha].sub.1] but not the [[Alpha].sub.2] and [[Alpha].sub.3] isoforms of [Na.sup.+]-[K.sup.+]-ATPase. Insulin increased the influx of [Rb.sup.+], used to trace [K.sup.+] entry, but did not modify the total intracellular content of [K.sup.+], [Rb.sup.+], and [Na.sup.+] over a 3-h incubation period. Ouabain increased intracellular [Na.sup.+] more rapidly in cells incubated with insulin, but this increase followed insulin stimulation of [Rb.sup.+] transport. Bumetanide did not prevent the increased [Na.sup.+] influx or stimulation of [Na.sup.+]-[K.sup.+]-ATPase. Stimulation of the [Na.sup.+]-[K.sup.+]-ATPase by insulin did not produce any measurable change in membrane potential. Insulin did not affect the affinity of the pump toward internal [Na.sup.+] or the number of membrane-bound [Na.sup.+]-[K.sup.+]-ATPases, as assessed by ouabain binding. By contrast, insulin slightly increased the affinity of [Na.sup.+]-[K.sup.+]-ATPase toward ouabain. Phorbol esters did not mimic insulin action on [Na.sup.+]-[K.sup.+]-ATPase and inhibited, rather than stimulated, [Rb.sup.+] transport. These results indicate that insulin increases the turnover rate of [Na.sup.+]-[K.sup.+]-ATPases of human fibroblasts without affecting their number on the plasma membrane or modifying their dependence on intracellular [[Na.sup.+]]. membrane transport; phorbol esters; membrane potential; rubidium transport

Chiu, Terence, and Enrique Rozengurt. PKD in intestinal epithelial cells: rapid activation by phorbol esters, LPA, and angiotensin through PKC. Am J Physiol Cell Physiol 280: C929-C942, 2001.--Protein kinase C (PKC) is implicated in the regulation of multiple important functions in intestinal epithelial cells, but the downstream signaling targets of PKCs in these cells remain poorly characterized. Here we report that treatment of normal rat intestinal cell lines IEC-6 and IEC-18 with phorbol 12,13-dibutyrate (PDBu) led to a rapid and striking PKC-dependent activation of protein kinase D (PKD; also known as PKC[micro]). Unlike conventional and novel PKCs, PKD did not undergo downregulation in response to prolonged (24 h) exposure of IEC-6 or IEC-18 cells to PDBu. PKD was also rapidly activated in these cells by lysophosphatidic acid (LPA) or angiotensin in a concentration-dependent fashion via a PKC-dependent pathway. [EC.sub.50] values were 0.1 [micro]M and 2 nM for LPA and angiotensin II, respectively. LPA-induced PKD activation was prevented selectively by treatment with pertussis toxin. PKD activation was tightly associated with an increase in PKD autophosphorylation at serine 916. Our results identify PKD as a novel early point of convergence and integration of [G.sub.i] and [G.sub.q] signaling in intestinal epithelial cells. IEC-18 cells; lysophosphatidic acid, protein kinase C downregulation; epithelial restitution; [G.sub.i] and [G.sub.q]

We describe a novel mechanism for protein kinase C regulation of axonal microtubule invasion of growth cones. Activation of PKC by phorbol esters resulted in a rapid, robust advance of distal microtubules (MTs) into the F-actin rich peripheral domain of growth cones, where they are normally excluded. In contrast, inhibition of PKC activity by bisindolylmaleimide and related compounds had no perceptible effect on growth cone motility, but completely blocked phorbol ester effects. Significantly, MT advance occurred despite continued retrograde F-actin flow--a process that normally inhibits MT advance. Polymer assembly was necessary for PKC-mediated MT advance since it was highly sensitive to a range of antagonists at concentrations that specifically interfere with microtubule dynamics. Biochemical evidence is presented that PKC activation promotes formation of a highly dynamic MT pool. Direct assessment of microtubule dynamics and translocation using the fluorescent speckle microscopy microtubule marking technique indicates PKC activation results in a nearly twofold increase in the typical lifetime of a MT growth episode, accompanied by a 1.7-fold increase and twofold decrease in rescue and catastrophe frequencies, respectively. No significant effects on instantaneous microtubule growth, shortening, or sliding rates (in either anterograde or retrograde directions) were observed. MTs also spent a greater percentage of time undergoing retrograde transport after PKC activation, despite overall MT advance. These results suggest that regulation of MT assembly by PKC may be an important factor in determining neurite outgrowth and regrowth rates and may play a role in other cellular processes dependent on directed MT advance. Key words: protein kinase C * microtubules * axon outgrowth * actin * growth cone

Schuette, Ralph, and Margot C. LaPointe. Phorbol ester stimulates cyclooxygenase-2 expression and prostanoid production in cardiac myocytes. Am J Physiol Heart Circ Physiol 279: H719-H725, 2000.--Phorbol-12-myristate-13-acetate (PMA) has been shown to induce hypertrophy of cardiac myocytes. The prostaglandin endoperoxide H synthase isoform 2 (cyclooxygenase-2, COX-2) has been associated with enhanced growth and/or proliferation of several types of cells. Thus we studied whether PMA induces COX-2 and prostanoid products [PGE.sub.2] and [PGF.sub.2[Alpha]], in neonatal ventricular myocytes and whether endogenous COX-2 products participate in their growth. In addition, we examined whether PMA affects interleukin-1[Beta] (IL-1[Beta]) stimulation of COX-2 and [PGE.sub.2] production. PMA (0.1 [micro]mol/l) stimulated growth, as indicated by a 1.6-fold increase in [[sup.3]H]leucine incorporation. PMA increased COX-2 protein levels 2.8-fold, [PGE.sub.2] 3.7-fold, and [PGF.sub.2[Alpha]], 2.9-fold. Inhibition of either p38 kinase or protein kinase C (PKC) prevented PMA-stimulated COX-2. Inhibition of COX-2 with either indomethacin or NS-398 had no effect on PMA-stimulated [[sup.3]H]leucine incorporation. Exogenous administration of [PGF.sub.2[Alpha]], but not [PGE.sub.2], stimulated protein synthesis. Treatment with IL-1[Beta] (5 ng/ml) increased COX-2 protein levels 42-fold, whereas cotreatment with IL-1[Beta] and PMA stimulated COX-2 protein only 32-fold. IL-1[Beta] did not affect control or PMA-stimulated protein synthesis. These findings indicate that: 1) PMA, acting through PKC and p38 kinase, enhances COX-2 expression, but chronic treatment with PMA partially inhibits IL-1[Beta] stimulation of COX-2; and 2) exogenous [PGF.sub.2[Alpha]], is involved in neonatal ventricular myocyte growth but endogenous COX-2 products are not. interleukin-1[Beta]; protein kinase C; neonatal cardiac myocytes; p38 kinase; hypertrophy