Increased expression of protein kinase C isoforms in heart failure due to myocardial infarction

The activities of cardiac protein kinase C (PKC) were examined in hemodynamically assessed rats subsequent to myocardial infarction (MI). Both Ca(2+)-dependent and Ca(2+)-independent PKC activities increased significantly in left ventricular (LV) and right ventricular (RV) homogenates at 1, 2, 4, and 8 wk after MI was induced. PKC activities were also increased in both LV and RV cytosolic and particulate fractions from 8-wk infarcted rats. The relative protein contents of PKC-alpha, -beta, -epsilon, and -zeta isozymes were significantly increased in LV homogenate, cytosolic (except PKC-alpha), and particulate fractions from the failing rats. On the other hand, the protein contents of PKC-alpha, -beta, and -epsilon isozymes, unlike the PKC-zeta isozyme, were increased in RV homogenate and cytosolic fractions, whereas the RV particulate fraction showed an increase in the PKC-alpha isozyme only. These changes in the LV and RV PKC activities and protein contents in the 8-wk infarcted animals were partially corrected by treatment with the angiotensin-converting enzyme inhibitor imidapril. No changes in protein kinase A activity and its protein content were seen in the 8-wk infarcted hearts. The results suggest that the increased PKC activity in cardiac dysfunction due to MI may be associated with an increase in the expression of PKC-alpha, -beta, and -epsilon isozymes, and the improvement of heart function in the infarcted animals by imidapril may be due to partial prevention of changes in PKC activity and isozyme contents.     

Losartan attenuates phospholipase C isozyme gene expression in hypertrophied hearts due to volume overload

Because the left ventricular (LV) hypertrophy due to volume overload induced by arteriovenous (AV) shunt was associated with an increase in phospholipase C (PLC) isozyme mRNA levels, PLC is considered to be involved in the development of cardiac hypertrophy. Since the renin-angiotensin system (RAS) is activated in cardiac hypertrophy, the role of RAS in the stimulation of PLC isozyme gene expression in hypertrophied heart was investigated by inducing AV shunt in Sprague-Dawley rats. The animals were treated with or without losartan (20 mg/kg, daily) for 3 days as well as 1, 2 and 4 weeks, and atria, right ventricle (RV) and LV were used for analysis. The increased muscle mass as well as the mRNA levels for PLC beta1 and beta3 in atria and RV, unlike PLC beta3 gene expression in LV, at 3 days of AVshunt were attenuated by losartan. The increased gene expression for PLC beta1 at 2 weeks in atria, at 1 and 4 weeks in RV, and at 2 and 4 weeks in LV was also depressed by losartan treatment. Likewise, the elevated mRNA levels for PLC beta3 in RV at 1 week and in LVat 4 weeks of cardiac hypertrophy were decreased by losartan. On the other hand, the increased levels of mRNA for PLC gamma1 in RV and LV at 2 and 4 weeks of inducing hypertrophy, unlike in atria at 4 weeks were not attenuated by losartan treatment. While the increased mRNA level for PLC delta1 in LV was reduced by losartan, gene expression for PLC delta1 was unaltered in atria and decreased in RV at 3 days of inducing AV shunt. These results suggest that changes in PLC isozyme gene expression were chamber specific and time-dependent upon inducing cardiac hypertrophy due to AV shunt. Furthermore, partial attenuation of the increased gene expression for some of the PLC isozymes and no effect of losartan on others indicate that both RAS dependent and independent mechanisms may be involved in hypertrophied hearts due to volume overload.     

Effects of moderate pressure overload cardiac hypertrophy on the distribution of creatine kinase isozymes

Myocardial activities and isozyme distributions of creatine kinase (CK) and lactate dehydrogenase (LDH) were measured in rats with moderate pressure overload hypertrophy. Three weeks after aortic banding, the ratio of left ventricular (LV) weight to body weight increased by 30%. Values for enzyme activity in the hypertrophied LV were compared to values for control rats as well as to the contralateral relatively unaffected right ventricle (RV). In rats with moderate LV hypertrophy, total CK activity was unchanged. The percent MB-CK increased significantly (p less than 0.01) only in the hypertrophied LV, from 13 +/- 1% to 19 +/- 1% of total CK, while the sum of MM and mitochondrial-CK decreased from 86 +/- 3 to 80 +/- 3% (p less than 0.01). LDH activity increased (p less than 0.05) only in the hypertrophied ventricle from a control of 2.90 +/- 0.13 to 3.21 +/- 0.13 IU/mg protein, while the ratio of LDH activity at high to low substrate increased from 0.12 +/- 0.02 to 0.14 +/- 0.02 (p less than 0.05). Thus, the development of moderate pressure overload hypertrophy in the LV is associated with normal levels of total CK, but the percentage of MB-CK increases selectively in the primarily affected ventricle. Also, total LDH and LDH activity at high to low substrate concentration increases significantly in LV hypertrophy.     

Expression of mitochondrial regulatory genes parallels respiratory capacity and contractile function in a rat model of hypoxia-induced right ventricular hypertrophy

Chronic hypobaric hypoxia (CHH) increases load on the right ventricle (RV) resulting in RV hypertrophy. We hypothesized that CHH elicits distinct responses, i.e., the hypertrophied RV, unlike the left ventricle (LV), displaying enhanced mitochondrial respiratory and contractile function. Wistar rats were exposed to 4 weeks CHH (11% O(2)) versus normoxic controls. RV/body weight ratio increased (P < 0.001 vs. control) while RV systolic and developed pressures were higher. However, LV systolic and developed pressures were significantly reduced. Mitochondrial O(2) consumption was sustained in the hypertrophied RV, ADP/O increased (P < 0.01 vs. control) and proton leak significantly decreased. Conversely, LV mitochondrial O(2) consumption was attenuated (P < 0.05 vs. control) and proton leak significantly increased. In parallel, expression of mitochondrial regulators was upregulated in the hypertrophied RV but not the LV. Our data show that the hypertrophied RV induces expression of mitochondrial regulatory genes linking respiratory capacity and enhanced efficiency to sustained contractile function.     

Effect of phorbol esters on protein kinase C-zeta.

Protein kinase C-zeta (PKC-zeta) is a member of the protein kinase C gene family which using in vitro preparations has been described as being resistant to activation by phorbol esters. PKC-zeta was found to be expressed in several cell types as an 80-kDa protein. In vitro translation of a full-length PKC-zeta construct also yielded as a primary translation product an 80-kDa protein. In the U937 cell, PKC-zeta was slightly more abundant in the cytosol than in the particulate fraction. Acute exposure of U937 cells to tetradecanoyl-phorbol-13-acetate (TPA), phorbol dibutyrate, mezerin, or diacylglycerol derivatives did not induce translocation of this isoform to the particulate fraction. Chronic exposure to 1 microM TPA failed to translocate or down-regulate PKC-zeta in U937, HL-60, COS, or HeLa-fibroblast fusion cells. To examine whether PKC-zeta was activated by TPA, PKC activity was evaluated in COS cells transiently over-expressing this isoform. In non-transfected cells, two peaks of phospholipid- and TPA-dependent kinase activity were observed. Eluting at a lower salt concentration was a peak of activity associated with PKC-alpha. PKC-zeta eluted with the second peak of activity and at a higher salt concentration. In transfected cells which expressed PKC-zeta at 4-10-fold over endogenous levels, there was only a slight increase in activity associated with the second peak. The activity and quantity of PKC-zeta did not strictly correlate. Treatment with TPA under conditions that did not alter PKC-zeta content abolished detection of the second peak of PKC activity eluting from the Mono Q column. Thus, PKC-zeta does not translocate or down-regulate in response to phorbol esters or diacylglycerol derivatives. However, for reasons discussed these studies do not resolve the issue of whether this isoform is activated by TPA.     

Protein kinase C-alpha and -beta play antagonistic roles in the differentiation process of THP-1 cells

The roles of protein kinase C (PKC) isoenzymes in the differentiation process of THP-1 cells are investigated. Inhibition of PKC by RO 31-8220 reduces the phagocytosis of latex particles and the release of superoxide, prostaglandin E(2) (PGE(2)), and tumour necrosis factor (TNF)-alpha. The proliferation of THP-1 cells is slightly enhanced by RO 31-8220. Stable transfection of THP-1 cells with asPKC-alpha, and incubation of THP-1 cells with antisense (as) PKC-alpha oligodeoxynucleotides reduces PKC-alpha levels and PKC activity. asPKC-alpha-transfected THP-1 cells show a decreased phagocytosis and a decreased release of superoxide, PGE(2) and TNF-alpha. The proliferation of asPKC-alpha-transfected THP-1 cells is enhanced. Stable transfection of THP-1 cells with asPKC-beta, and incubation of THP-1 cells with asPKC-beta oligodeoxynucleotides, reduces PKC-beta levels and PKC activity. asPKC-beta-transfected THP-1 cells show a decreased phagocytosis, a decreased TNF-alpha release, and a decreased proliferation. However, no difference is measured in the release of superoxide and PGE(2). These results suggest that: (1) PKC-alpha but not PKC-beta is involved in the release of superoxide and PGE(2); (2) TNF-alpha release and the phagocytosis of latex particles are mediated by PKC-alpha, PKC-beta, and other PKC isoenzymes; and (3) PKC-alpha and PKC-beta play antagonistic roles in the differentiation process of THP-1 cells. PKC-alpha promotes the differentiation process of THP-1 cells, PKC-beta retards the differentiation of THP-1 cells into macrophage-like cells.     

Protein kinase C-gamma is present in adriamycin resistant HL-60 leukemia cells

The isoform pattern of protein kinase C (PKC) was examined in wild-type and Adriamycin-resistant (HL-60/AR) HL-60 leukemia cells. Analyses were carried out by immunoblotting with mouse monoclonal antibodies against PKC-alpha and PKC-beta and a rabbit polyclonal antibody against the variable (V3) region of PKC-gamma. HL-60/AR cells contained an equivalent level of PKC-alpha and a lower amount of PKC-beta than HL-60 cells. In contrast, only HL-60/AR cells contained PKC-gamma. These results indicate that the regulation of this family of isoenzymes is altered in drug-resistant cells.     

Right and left ventricular function after chronic pulmonary artery banding in rats assessed with biventricular pressure-volume loops

In many patients with congenital heart disease, the right ventricle (RV) is subjected to abnormal loading conditions. To better understand the state of compensated RV hypertrophy, which could eventually progress to decompensation, we studied the effects of RV pressure overload in rats. In the present study, we report the biventricular adaptation to 6 wk of pulmonary artery banding (PAB). PAB resulted in an RV pressure overload to approximately 60% of systemic level and a twofold increase in RV mass (P < 0.01). Systemic hemodynamic parameters were not altered, and overt signs of heart failure were absent. Load-independent measures of ventricular function (end-systolic pressure-volume relation, preload recruitable stroke work relation, maximum first time derivative of pressure divided by end-diastolic volume), assessed by means of pressure-volume (PV) loops, demonstrated a two- to threefold increase in RV contractility under baseline conditions in PAB rats. RV contractility increased in response to dobutamine stimulation (2.5 microg.kg(-1).min(-1)) both in PAB and sham-operated rats in a similar fashion, indicating preserved RV contractile reserve in PAB rats. Left ventricular (LV) contractility at baseline was unaffected in PAB rats, although LV volume in PAB rats was slightly decreased. LV contractility increased in response to dobutamine (2.5 microg.kg(-1).min(-1)), both in PAB and sham rats, whereas the response to a higher dose of dobutamine (5 microg.kg(-1).min(-1)) was blunted in PAB rats. RV pressure overload (6 wk) in rats resulted in a state of compensated RV hypertrophy with preserved RV contractile reserve, whereas LV contractile state at baseline was not affected. Furthermore, this study demonstrates the feasibility of performing biventricular PV-loop measurements in rats.     

Captopril fails to reverse hypertrophy of the left ventricle induced by aortic insufficiency in rabbits

Angiotensin converting enzyme (ACE) inhibition has been reported to induce regression of hypertrophy in several models of hemodynamic pressure overload. The aim of the present study was to determine whether the ACE inhibitor captopril can reduce hypertrophy of the left ventricle induced by a chronic volume overload and modify collagen composition of the hypertrophied myocardium. Rabbits with four months lasting aortic insufficiency were divided into two groups: treated with captopril (20 mg/kg/day) for five weeks and treated with placebo. The respective control groups were represented by sham-operated animals. Aortic insufficiency induced a decrease of diastolic pressure, an increase of systolic and pulse pressure, hypertrophy of the left and right ventricle, and an increase of hydroxyproline content in the left ventricle without a change of hydroxyproline concentrations in either ventricle. Captopril treatment further enhanced pulse pressure by decreasing diastolic blood pressure. Hypertrophy of the left ventricle, hydroxyproline content and concentration in both ventricles were unaffected by captopril treatment. It is concluded that ACE inhibition did not reverse the left ventricular hypertrophy developed as a result of overload induced by aortic insufficiency. We suggest that mechanisms different from activation of the renin-angiotensin system may play a decisive role in the maintenance of hypertrophy in this particular model of volume hemodynamic overload.     

The expression of mRNA of cytokines and of extracellular matrix proteins in triiodothyronine-treated rat hearts

In various models of cardiac hypertrophy, e.g. treatment of rats with norepinephrine infusion or pressure overload, increased expression of cytokines together with increase in extracellular matrix proteins (ECMP) was reported. In this study the effect of triiodothyronine (T3) on the expression of mRNA for cytokines and ECMP was investigated. Female Sprague-Dawley rats were treated daily with T3 in a dose of 0.2 mg.kg^–1 of body weight s.c. Changes in the left (LV) and right (RV) ventricular function were measured 6, 24, 48, 72 h and 7 and 14 days after the first T3-injection using Millar ultraminiature pressure catheter transducers. RNA was isolated from LV and RV tissue, and the expression of cytokines and ECMP was measured using the ribonuclease protection assay. T3-treatment induced a significant increase in LV dP/dt_max and RV dP/dt_max, (p < 0.05) 24 h after the first injection of T3 together with an increase in heart rate (p < 0.01). The RV systolic pressure increased 48 h after the first T3 injection, whereas the LV systolic pressure remained unchanged. After 48 h the heart weight to body weight ratio was increased (p< 0.01). Hypertrophy of the RV was more prominent than that of the LV (155.9 vs. 137.7%).In all groups the expression of mRNA for interleukins (IL) IL-6, IL-1, IL-1 and tumour necrosis factor (TNF)- in both ventricles did not change (p > 0.05). There was a significant increase in the mRNA for colligin 24 h after the T3 injection in both LV (p < 0.01) and RV (p< 0.05). This was followed by an increase in the mRNA for collagen I and III 72 h after the first T3-dose (p < 0.05 in RV; p < 0.01 in LV). At this point, the mRNA for tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) was increased (p < 0.01) in the LV only. Moreover, after 7 days also the mRNA for matrix metalloproteinase (MMP)-2 increased (p < 0.01) in the LV. Both, TIMP-2 and MMP-2 were increased in the RV only after 14 days (p < 0.05). The gelatinase activity of MMP-2, however, was unchanged in both ventricles. The T3-induced cardiac hypertrophy was not accompanied by fibrosis as measured by the Sirius red staining after 14-days of T3-treatment. The moderate increase in mRNA for ECMP and MMP may be attributed more to the increasing mass of the ventricles with the accompanying remodelling of the ECM than to increased fibrosis.     

Antioxidant and oxidative stress changes in experimental cor pulmonale

Although right heart failure (RHF) contributes to 20% of all cardiovascular complications, most of the information available on RHF in general is based on the experiences with left heart failure. This study on RHF investigates changes in antioxidants and oxidative stress which are suggested to play a role in the transition from hypertrophy to failure. RHF subsequent to pulmonary hypertension was produced in rats by a single injection of monocrotaline (MCT, 60 mg/kg, i.p.). Based on hemodynamic, clinical and histopathologic observations, the animals were grouped in three functional stages at 1-, 2- and 6-week post-injection periods. In the 1-week group, RV pressure overload and hypertrophy, and a mild increase in antioxidant enzymes was seen. In the 2-week group, compensated HF, a significant increase in antioxidant enzymes, an increase in septal (IVS) wall thickness and leftward displacement of IVS without change in LV free wall were seen. In the 6-week group, lung and liver congestion, RVF and dilation, a decrease in antioxidant enzyme activities, increase in lipid peroxidation and severe bulging of the IVS into the left ventricle were seen. These changes in the hemodynamic, biochemical and histopathologic characteristics suggest that in early stages of MCT-induced pulmonary hypertension at 1 and 2 weeks, RV hypertrophy was accompanied by sustained hemodynamic function and an increase in antioxidant reserve. In the later stage at 6 weeks, clinical RHF was associated with abnormalities of the right heart systolic and diastolic function along with a decrease in antioxidant reserve. These biphasic changes in RV antioxidant enzymes, i.e. an increase during hypertrophy and a decrease in failure may suggest a role of oxidative stress in the pathogenesis of right ventricular dysfunction.     

Induction of heat shock protein 72 in the failing heart is attenuated after an exposure to heat shock

Induction of heat shock protein (Hsp) 72 in the right ventricular muscle of the rat with heart failure following acute myocardial infarction (AMI) was examined. AMI was induced by the left coronary artery ligation (CAL). The animals at the 8th, but not 2nd, week after CAL revealed a decrease in cardiac output index (COI), suggesting that heart failure had developed by 8 weeks after CAL. Increases in the right ventricular developed pressure and the ratios of right ventricle/body weight and lung/body weight at the 2nd and 8th weeks showed the development of the right ventricular hypertrophy. After measurement of hemodynamic parameters, the hearts isolated from animals at the 2nd and 8th weeks after CAL (2w- and 8w-CAL hearts, respectively) were perfused and subjected to heat shock (at 42 degrees C, for 15 min) followed by 6-h perfusion. At the end of perfusion, Hsp72 content in the left ventricle without infarct area (viable LV) and the right ventricle (RV) was determined by the Western immunoblotting method. The production of myocardial Hsp72 in the viable LV and RV of the 2w-CAL heart increased after an exposure to heat shock. In contrast, induction of Hsp72 in the viable LV and RV of the 8w-CAL heart was blunted. The results suggest that the development of heart failure following AMI may result in a decrease in the ability for Hsp72 induction not only in the viable LV but also in the RV, leading to contractile dysfunction of the heart.     

Protein kinase C isozymes and their selectivity towards ruboxistaurin

Protein kinase C (PKC) isozymes are an important class of enzymes in cell signaling and as drug targets. They are involved in specific pathways and have selectivity towards certain ligands, despite their high sequence similarities. Ruboxistaurin is a specific inhibitor of PKC-beta. To understand the molecular determinants for the selectivity of ruboxistaurin, we derived the three-dimensional structures of the kinase domains of PKC-alpha, -betaI, and -zeta using homology modeling. Several binding orientations of ruboxistaurin in the binding sites of these PKC catalytic domains were analyzed, and a putative alternative binding site for PKC-zeta was identified in its kinase domain. The calculated free energy of binding correlates well with the IC(50) of the inhibitor against each PKC isozyme. A residue-based energy decomposition analysis attributed the binding free energy to several key residues in the catalytic sites of these enzymes, revealing potential protein-ligand interactions responsible for ligand binding. The contiguous binding site revealed in the catalytic domain of PKC-zeta provides avenues for selective drug design. The details of structural nuances for specific inhibition of PKC isozymes are presented in the context of the three-dimensional structures of this important class of enzymes.     

Antioxidant and oxidative stress changes in experimental cor pulmonale

Although right heart failure (RHF) contributes to 20% of all cardiovascular complications, most of the information available on RHF in general is based on the experiences with left heart failure. This study on RHF investigates changes in antioxidants and oxidative stress which are suggested to play a role in the transition from hypertrophy to failure. RHF subsequent to pulmonary hypertension was produced in rats by a single injection of monocrotaline (MCT, 60 mg/kg, i.p.). Based on hemodynamic, clinical and histopathologic observations, the animals were grouped in three functional stages at 1-, 2- and 6-week post-injection periods. In the 1-week group, RV pressure overload and hypertrophy, and a mild increase in antioxidant enzymes was seen. In the 2-week group, compensated HF, a significant increase in antioxidant enzymes, an increase in septal (IVS) wall thickness and leftward displacement of IVS without change in LV free wall were seen. In the 6-week group, lung and liver congestion, RVF and dilation, a decrease in antioxidant enzyme activities, increase in lipid peroxidation and severe bulging of the IVS into the left ventricle were seen. These changes in the hemodynamic, biochemical and histopathologic characteristics suggest that in early stages of MCT-induced pulmonary hypertension at 1 and 2 weeks, RV hypertrophy was accompanied by sustained hemodynamic function and an increase in antioxidant reserve. In the later stage at 6 weeks, clinical RHF was associated with abnormalities of the right heart systolic and diastolic function along with a decrease in antioxidant reserve. These biphasic changes in RV antioxidant enzymes, i.e. an increase during hypertrophy and a decrease in failure may suggest a role of oxidative stress in the pathogenesis of right ventricular dysfunction.     

Expression of protein kinase C genes in hemopoietic cells is cell-type- and B cell-differentiation stage specific

We have studied the expression of mRNA encoding all known protein kinase C (PKC) isozymes (alpha, beta, gamma, delta, epsilon, zeta, and eta) in murine tumor cell lines that exemplify hemopoietic cells arrested at different stages of development as well as in normal hemopoietic cells. We demonstrate that some of the isozymes, PKC-alpha, -beta, and -eta, are differentially expressed in different lineages. PKC-alpha and -beta generally are not detectable in myeloid cell lines, where PKC-delta is the predominant isoform. Both PKC-alpha and -beta are abundant in most T and B lymphocytic lines, but steady state levels of PKC-beta mRNA are lowest in plasma cell tumors, which exemplify the terminally differentiated B lymphocyte. In contrast, the levels of PKC-alpha mRNA remain high in plasma cell tumors, and a novel, 2.5-kb PKC-alpha mRNA gains prominence. PKC-eta mRNA is the major PKC isoform expressed in T lymphocytes, but it also is highly abundant in some myeloid lines. PKC-delta is expressed at high levels in all the lines we studied, whereas PKC-epsilon and -zeta are found in most cells but only at rather low levels. Analysis of myeloid clones derived from bipotential B lineage progenitor cell lines suggests that the B cell phenotype is associated with the expression of PKC-alpha. The close correlation of protein levels with mRNA levels indicates that PKC expression in hemopoietic cells is mainly regulated at the level of mRNA. The lineage- and differentiation stage-specific patterns of PKC-isozyme expression presented here suggest the involvement of specific PKC isozymes in differentiation as well as lineage determination of hemopoietic cells.     

Gbetagamma-dependent phosphoinositide 3-kinase activation in hearts with in vivo pressure overload hypertrophy

Activation of phosphoinositide 3-kinases is coupled to both phosphotyrosine/growth factor and G protein-coupled receptors. We explored the role of phosphoinositide 3-kinase activation in myocardium during in vivo pressure overload hypertrophy in mice. Cytosolic extracts from wild type hypertrophied hearts showed a selective increase in the phosphoinositide 3-kinase gamma isoform. To address the role of G protein-coupled receptor-mediated activation of phosphoinositide 3-kinase, we used transgenic mice with cardiac-specific overexpression of a Gbetagamma sequestering peptide. Extracts from hypertrophied transgenic hearts showed complete loss of phosphoinositide 3-kinase activation, indicating a Gbetagamma-dependent process. To determine the class of G proteins that contribute Gbetagamma dimers for in vivo phosphoinositide 3-kinase activation, two strategies were used: 1) transgenic mice with cardiac-specific overexpression of a G(q) inhibitor peptide and 2) pertussis toxin treatment prior to pressure overload in wild type mice. Pressure overloaded G(q) inhibitor transgenic mice showed a complete absence of phosphoinositide 3-kinase activation, whereas pretreatment with pertussis toxin showed robust phosphoinositide 3-kinase activation. Taken together, these data demonstrate that activation of the phosphoinositide 3-kinase during in vivo pressure overload hypertrophy is Gbetagamma-dependent and the Gbetagamma dimers arise from stimulation of G(q)-coupled receptors.     

Cationic porphyrins: novel delivery vehicles for antisense oligodeoxynucleotides.

Cationic porphyrins form stable complexes with oligodeoxynucleotides. To evaluate delivery, we used a 20mer phosphorothioate oligomer (Isis 3521) targeted to the 3'-untranslated region of the PKC-alpha mRNA, and complexed it with porphyrin. The expression of PKC-alpha protein and mRNA in T24 bladder carcinoma cells was reduced by approximately 80 +/- 10% at a concentration of oligomer of 3 microM, and 9 microM porphyrin. The expression of PKC-beta1, -delta and -straightepsilon isoforms was unaffected by this treatment, but elimination of PKC-zeta protein and mRNA were observed. However, treatment with the porphyrin complex of Isis 3522, an oligomer which is directed at the 5' coding region of the PKC-alpha mRNA, was equally effective as Isis 3521 with respect to PKC-alpha, but did not affect PKC-zeta protein or mRNA levels. Since Isis 3521 has an 11-base region of complementarity with the PKC-zeta mRNA, wheras Isis 3522 has only a 4-base region, the effect of Isis 3521 on PKC-zeta protein and mRNA expression may be due to irrelevant cleavage. Depending upon the desired application, this new strategy may offer several advantages over other methods of antisense oligodeoxynucleotide delivery including efficiency, stability, solubility, relatively low toxicity and serum compatibility. Porphyrins may thus be a potentially useful delivery vehicle for antisense therapeutics and/or target validation.     

Chronic PKC-beta activation in HT-29 Cl.19a colonocytes prevents cAMP-mediated ion secretion by inhibiting apical membrane current generation

We investigated the effects of PKC-stimulating 12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPA) and phorbol 12-myristate 13-acetate (PMA) phorbol esters on cAMP-dependent, forskolin (FSK)-stimulated, short-circuit Cl- current (ISC-cAMP) generation by colonocyte monolayers. These agonists elicited different actions depending on their dose and incubation time; PMA effects at the onset (<5 min) were independent of cAMP agonist and were characterized by transient anion-dependent transcellular and apical membrane ISC generation. DOPPA failed to elicit similar responses. Whereas chronic (24 h) exposure to both agents inhibited FSK-stimulated transcellular and apical membrane ISC-cAMP, the effects of DOPPA were more complex: this conventional PKC-beta-specific agonist also stimulated Ba2+-sensitive basolateral membrane-dependent facilitation of transcellular ISC-cAMP. PMA did not elicit a similar phenomenon. Prolonged exposure to high-dose PMA but not DOPPA led to apical membrane ISC-cAMP recovery. Changes in PKC alpha-, beta1-, gamma-, and epsilon-isoform membrane partitioning and expression correlated with these findings. PMA-induced transcellular ISC correlated with PKC-alpha membrane association, whereas low doses of both agents inhibited transcellular and apical membrane ISC-cAMP, increased PKC-beta1, decreased PKC-beta2 membrane association, and caused reciprocal changes in isoform mass. During the apical membrane ISC-cAMP recovery after prolonged high-dose PMA exposure, an almost-complete depletion of cellular PKC-beta1 and a significant reduction in PKC-epsilon mass occurred. Thus activated PKC-beta1 and/or PKC-epsilon prevented, whereas activated PKC-alpha facilitated, apical membrane ISC-cAMP. PKC-beta-dependent augmentation of transcellular ISC-cAMP at the level of the basolateral membrane demonstrated that transport events with geographically distinct subcellular membranes can be independently regulated by the PKC beta-isoform.     

Protein Kinase-zeta inhibits collagen I-dependent and anchorage-independent growth and enhances apoptosis of human Caco-2 cells

Colonic carcinogenesis is accompanied by abnormalities in multiple signal transduction components, including alterations in protein kinase C (PKC). The expression level of PKC-zeta, an atypical PKC isoform, increases from the crypt base to the luminal surface and parallels crypt cell differentiation in normal colon. In prior studies in the azoxymethane model of colon cancer, we showed that PKC-zeta was down-regulated in rat colonic tumors. In this study, we showed that PKC-zeta is expressed predominantly in colonic epithelial and not stromal cells, and loss of PKC-zeta occurs as early as the adenoma stage in human colonic carcinogenesis. To assess the regulation of growth and differentiation by PKC-zeta, we altered this isoform in human Caco-2 colon cancer cells using stable constitutive or inducible expression vectors, specific peptide inhibitors or small interfering RNA. In ecdysone-regulated transfectants grown on collagen I, ponasterone A significantly induced PKC-zeta expression to 135% of empty vector cells, but did not alter nontargeted PKC isoforms. This up-regulation was accompanied by a 2-fold increase in basal and 4-fold increase in insulin-stimulated PKC-zeta biochemical activity. Furthermore, PKC-zeta up-regulation caused >50% inhibition of cell proliferation on collagen I (P < 0.05). Increased PKC-zeta also significantly enhanced Caco-2 cell differentiation, nearly doubling alkaline phosphatase activity, while inducing a 3-fold increase in the rate of apoptosis (P < 0.05). In contrast, knockdown of this isoform by small interfering RNA or kinase inhibition by myristoylated pseudosubstrate significantly and dose-dependently increased Caco-2 cell growth on collagen I. In transformation assays, constitutively up-regulated wild-type PKC-zeta significantly inhibited Caco-2 cell growth in soft agar, whereas a kinase-dead mutant caused a 3-fold increase in soft agar growth (P < 0.05). Taken together, these studies indicate that PKC-zeta inhibits colon cancer cell growth and enhances differentiation and apoptosis, while inhibiting the transformed phenotype of these cells. The observed down-regulation of this growth-suppressing PKC isoform in colonic carcinogenesis would be predicted to contribute to tumorigenesis.