PKC delta-isoform translocation and enhancement of tonic contractions of gastrointestinal smooth muscle

PKC is involved in mediating the tonic component of gastrointestinal smooth muscle contraction in response to stimulation by agonists for G protein-coupled receptors. Here, we present pharmacological and immunohistochemical evidence indicating that a member of the novel PKC isoforms, PKC-delta, is involved in maintaining muscarinic receptor-coupled tonic contractions of the guinea pig ileum. The tonic component of carbachol-evoked contractions was enhanced by an activator of conventional and novel PKCs, phorbol 12,13-dibutyrate (PDBu; 200 nM or 1 microM), and by an activator of novel PKCs, ingenol 3,20-dibenzoate (IDB; 100 or 500 nM). Enhancement was unaffected by concentrations of bisindolylmaleimide I (BIM-I; 22 nM) that block conventional PKCs or by a PKC-epsilon-specific inhibitor peptide but was attenuated by higher doses of BIM-I (2.2 microM). Relevant proteins were localized at a cellular and subcellular level using confocal analysis. Immunohistochemical staining of the ileum showed that PKC-delta was exclusively expressed in smooth muscles distributed throughout the layers of the gut wall. PKC-epsilon immunoreactivity was prominent in enteric neurons but was largely absent from smooth muscle of the muscularis externa. Treatment with PDBu, IDB, or carbachol resulted in a time- and concentration-dependent translocation of PKC-delta from the cytoplasm to filamentous structures within smooth muscle cells. These were parallel to, but distinct from, actin filaments. The translocation of PKC-delta in response to carbachol was significantly reduced by scopolamine or calphostin C. The present study indicates that the tonic carbachol-induced contraction of the guinea pig ileum is mediated through a novel PKC, probably PKC-delta.     

PKC-delta and -epsilon regulate NF-kappaB activation induced by cholecystokinin and TNF-alpha in pancreatic acinar cells

Although NF-kappaB plays an important role in pancreatitis, mechanisms underlying its activation remain unclear. We investigated the signaling pathways mediating NF-kappaB activation in pancreatic acinar cells induced by high-dose cholecystokinin-8 (CCK-8), which causes pancreatitis in rodent models, and TNF-alpha, which contributes to inflammatory responses of pancreatitis, especially the role of PKC isoforms. We determined subcellular distribution and kinase activities of PKC isoforms and NF-kappaB activation in dispersed rat pancreatic acini. We applied isoform-specific, cell-permeable peptide inhibitors to assess the role of individual PKC isoforms in NF-kappaB activation. Both CCK-8 and TNF-alpha activated the novel isoforms PKC-delta and -epsilon and the atypical isoform PKC-zeta but not the conventional isoform PKC-alpha. Inhibition of the novel PKC isoforms but not the conventional or the atypical isoform resulted in the prevention of NF-kappaB activation induced by CCK-8 and TNF-alpha. NF-kappaB activation by CCK-8 and TNF-alpha required translocation but not tyrosine phosphorylation of PKC-delta. Activation of PKC-delta, PKC-epsilon, and NF-kappaB with CCK-8 involved both phosphatidylinositol-specific PLC and phosphatidylcholine (PC)-specific PLC, whereas with TNF-alpha they only required PC-specific PLC for activation. Results indicate that CCK-8 and TNF-alpha initiate NF-kappaB activation by different PLC pathways that converge at the novel PKCs (delta and epsilon) to mediate NF-kappaB activation in pancreatic acinar cells. These findings suggest a key role for the novel PKCs in pancreatitis.     

Protein kinase C modulates inactivation of Kv3.3 channels

Modulation of some Kv3 family potassium channels by protein kinase C (PKC) regulates their amplitude and kinetics and adjusts firing patterns of auditory neurons in response to stimulation. Nevertheless, little is known about the modulation of Kv3.3, a channel that is widely expressed throughout the nervous system and is the dominant Kv3 family member in auditory brainstem. We have cloned the cDNA for the Kv3.3 channel from mouse brain and have expressed it in a mammalian cell line and in Xenopus oocytes to characterize its biophysical properties and modulation by PKC. Kv3.3 currents activate at positive voltages and undergo inactivation with time constants of 150-250 ms. Activators of PKC increased current amplitude and removed inactivation of Kv3.3 currents, and a specific PKC pseudosubstrate inhibitor peptide prevented the effects of the activators. Elimination of the first 78 amino acids of the N terminus of Kv3.3 produced noninactivating currents suggesting that PKC modulates N-type inactivation, potentially by phosphorylation of sites in this region. To identify potential phosphorylation sites, we investigated the response of channels in which serines in this N-terminal domain were subjected to mutagenesis. Our results suggest that serines at positions 3 and 9 are potential PKC phosphorylation sites. Computer simulations of model neurons suggest that phosphorylation of Kv3.3 by PKC may allow neurons to maintain action potential height during stimulation at high frequencies, and may therefore contribute to stimulus-induced changes in the intrinsic excitability of neurons such as those of the auditory brainstem.     

Protein kinase C-epsilon-null mice have decreased hypoxic pulmonary vasoconstriction

PKC contributes to regulation of pulmonary vascular reactivity in response to hypoxia. The role of individual PKC isozymes is less clear. We used a knockout (null, -/-) mouse to test the hypothesis that PKC-epsilon is important in acute hypoxic pulmonary vasoconstriction (HPV). We asked whether deletion of PKC-epsilon would decrease acute HPV in adult C57BL6xSV129 mice. In isolated, salt solution-perfused lung, reactivity to acute hypoxic challenges (0% and 3% O(2)) was compared with responses to angiotensin II (ANG II) and KCl. PKC-epsilon -/- mice had decreased HPV, whereas responses to ANG II and KCl were preserved. Inhibition of nitric oxide synthase (NOS) with nitro-l-arginine augmented HPV in PKC-epsilon +/+ but not -/- mice. Inhibition of Ca(2+)-gated K(+) channels (K(Ca)) with charybdotoxin and apamin did not enhance HPV in -/- mice relative to wild-type (+/+) controls. In contrast, the voltage-gated K(+) channel (K(V)) antagonist 4-aminopyridine increased the response of -/- mice beyond that of +/+ mice. This suggested that increased K(V) channel expression could contribute to blunted HPV in PKC-epsilon -/- mice. Therefore, expression of the O(2)-sensitive K(V) channel subunit Kv3.1b (100-kDa glycosylated form and 70-kDa core protein) was compared in whole lung and pulmonary artery smooth muscle cell (PASMC) lysates from +/+ and -/- mice. A subtle increase in Kv3.1b was detected in -/- vs. +/+ whole lung lysates. A much greater rise in Kv3.1b expression was found in -/- vs. +/+ PASMC. Thus deletion of PKC-epsilon blunts murine HPV. The decreased response could not be attributed to a general loss in vasoreactivity or derangements in NOS or K(Ca) channel activity. Instead, the absence of PKC-epsilon allows increased expression of K(V) channels (like Kv3.1b) to occur in PASMC, which likely contributes to decreased HPV.     

Deficiency of neural recognition molecule NB-2 affects the development of glutamatergic auditory pathways from the ventral cochlear nucleus to the superior olivary complex in mouse

Neural recognition molecule NB-2/contactin 5 is expressed transiently during the first postnatal week in glutamatergic neurons of the central auditory system. Here, we investigated the effect of NB-2 deficiency on the auditory brainstem in mouse. While almost all principal neurons are wrapped with the calyces of Held in the medial nucleus of the trapezoid body (MNTB) in wild type, 8% of principal neurons in NB-2 knockout (KO) mice lack the calyces of Held at postnatal day (P) 6. At P10 and P15, apoptotic principal neurons were detected in NB-2 KO mice, but not in wild type. Apoptotic cells were also increased in the ventral cochlear nucleus (VCN) of NB-2 KO mice, which contains bushy neurons projecting to the MNTB and the lateral superior olive (LSO). At the age of 1 month, the number of principal neurons in the MNTB and of glutamatergic synapses in the LSO was reduced in NB-2 KO mice. Finally, interpeak latencies for auditory brainstem response waves II-III and III-IV were significantly increased in NB-2 KO mice. Together, these findings suggest that NB-2 deficiency causes a deficit in synapse formation and then induces apoptosis in MNTB and VCN neurons, affecting auditory brainstem function.     

EphB signaling regulates target innervation in the developing and deafferented auditory brainstem

Precision in auditory brainstem connectivity underlies sound localization. Cochlear activity is transmitted to the ventral cochlear nucleus (VCN) in the mammalian brainstem via the auditory nerve. VCN globular bushy cells project to the contralateral medial nucleus of the trapezoid body (MNTB), where specialized axons terminals, the calyces of Held, encapsulate MNTB principal neurons. The VCN-MNTB pathway is an essential component of the circuitry used to compute interaural intensity differences that are used for localizing sounds. When input from one ear is removed during early postnatal development, auditory brainstem circuitry displays robust anatomical plasticity. The molecular mechanisms that control the development of auditory brainstem circuitry and the developmental plasticity of these pathways are poorly understood. In this study we examined the role of EphB signaling in the development of the VCN-MNTB projection and in the reorganization of this pathway after unilateral deafferentation. We found that EphB2 and EphB3 reverse signaling are critical for the normal development of the projection from VCN to MNTB, but that successful circuit assembly most likely relies upon the coordinated function of many EphB proteins. We have also found that ephrin-B reverse signaling repels induced projections to the ipsilateral MNTB after unilateral deafferentation, suggesting that similar mechanisms regulate these two processes.     

Protein kinase C inhibitors alter neurotensin receptor binding and function in prostate cancer PC3 cells

Prostate cancer PC3 cells expressed constitutive protein kinase C (PKC) activity that under basal conditions suppressed neurotensin (NT) receptor function. The endogenous PKC activity, assessed using a cell-based PKC substrate phosphorylation assay, was diminished by PKC inhibitors and enhanced by phorbol myristic acid (PMA). Accordingly, PKC inhibitors (staurosporine, Go-6976, Go-6983, Ro-318220, BIS-1, chelerythrine, rottlerin, quercetin) enhanced NT receptor binding and NT-induced inositol phosphate (IP) formation. In contrast, PMA inhibited these functions. The cells expressed conventional PKCs (, βI) and novel PKCs (δ, ε), and the effects of PKC inhibitors on NT binding were blocked by PKC downregulation. The inhibition of NT binding by PMA was enhanced by okadaic acid and blocked by PKC inhibitors. However, when some PKC inhibitors (rottlerin, BIS-1, Ro-318220, Go-69830, quercetin) were used at higher concentrations (> 2 μM), they had a different effect characterized by a dramatic increase in NT binding and an inhibition of NT-induced IP formation. The specificity of the agents implicated novel PKCs in this response and indeed, the inhibition of NT-induced IP formation was reproduced by PKCδ or PKCε knockdown. The inhibition of IP formation appeared to be specific to NT since it was not observed in response to bombesin. Scatchard analyses indicated that the PKC-directed agents modulated NT receptor affinity, not receptor number or receptor internalization. These findings suggest that PKC participates in heterologous regulation of NT receptor function by two mechanisms: a) — conventional PKCs inhibit NT receptor binding and signaling; and b) — novel PKCs maintain the ability of NT to stimulate PLC. Since NT can activate PKC upon binding to its receptor, it is possible that NT receptor is also subject to homologous regulation by PKC.     

The linoleic acid derivative DCP-LA selectively activates PKC-epsilon, possibly binding to the phosphatidylserine binding site

This study examined the effect of 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), a newly synthesized linoleic acid derivative with cyclopropane rings instead of cis-double bonds, on protein kinase C (PKC) activity. In the in situ PKC assay with reverse-phase high-performance liquid chromatography, DCP-LA significantly activated PKC in PC-12 cells in a concentration-dependent (10 nM-100 microM) manner, with the maximal effect at 100 nM, and the DCP-LA effect was blocked by GF109203X, a PKC inhibitor, or a selective inhibitor peptide of the novel PKC isozyme PKC-epsilon. Furthermore, DCP-LA activated PKC in HEK-293 cells that was inhibited by the small, interfering RNA against PKC-epsilon. In the cell-free PKC assay, of the nine isozymes examined here, DCP-LA most strongly activated PKC-epsilon, with >7-fold potency over other PKC isozymes, in the absence of dioleoyl-phosphatidylserine and 1,2-dioleoyl-sn-glycerol; instead, the DCP-LA action was inhibited by dioleoyl-phosphatidylserine. DCP-LA also activated PKC-gamma, a conventional PKC, but to a much lesser extent compared with that for PKC-epsilon, by a mechanism distinct from PKC-epsilon activation. Thus, DCP-LA serves as a selective activator of PKC-epsilon, possibly by binding to the phosphatidylserine binding site on PKC-epsilon. These results may provide fresh insight into lipid signaling in PKC activation.     

Bryostatin-1 attenuates TNF-induced epithelial barrier dysfunction: role of novel PKC isozymes

Tumor necrosis factor (TNF) increases epithelial permeability in many model systems. Protein kinase C (PKC) isozymes regulate epithelial barrier function and alter ligand-receptor interactions. We sought to define the impact of PKC on TNF-induced barrier dysfunction in T84 intestinal epithelia. TNF induced a dose- and time-dependent fall in transepithelial electrical resistance (TER) and an increase in [(3)H]mannitol flux. The TNF-induced fall in TER was not PKC mediated but was prevented by pretreatment with bryostatin-1, a PKC agonist. As demonstrated by a pattern of sensitivity to pharmacological inhibitors of PKC, this epithelial barrier preservation was mediated by novel PKC isozymes. Bryostatin-1 reduced TNF receptor (TNF-R1) surface availability, as demonstrated by radiolabeled TNF binding and cell surface biotinylation assays, and increased TNF-R1 receptor shedding. The pattern of sensitivity to isozyme-selective PKC inhibitors suggested that these effects were mediated by activation of PKC-epsilon. In addition, after bryostatin-1 treatment, PKC-delta and TNF-R1 became associated, as determined by mutual coimmunoprecipitation assay, which has been shown to lead to receptor desensitization in neutrophils. TNF-induced barrier dysfunction occurs independently of PKC, but selective modulation of novel PKC isozymes may regulate TNF-R1 signaling.     

PKC-epsilon regulation of extracellular signal-regulated kinase: a potential role in phenylephrine-induced cardiocyte growth

Hypertrophic growth of cardiac muscle is dependent on activation of the PKC-epsilon isoform. To define the effectors of PKC-epsilon involved in growth regulation, recombinant adenoviruses were used to overexpress either wild-type PKC-epsilon (PKC-epsilon/WT) or dominant negative PKC-epsilon (PKC-epsilon/DN) in neonatal rat cardiocytes. PKC-epsilon/DN inhibited acute activation of PKC-epsilon produced in response to phorbol ester and reduced ERK1/2 activity as measured by the phosphorylation of p42 and p44 isoforms. The inhibitory effects were specific to PKC-epsilon because PKC-epsilon/DN did not prevent translocation of either PKC-alpha or PKC-delta. Overexpression of PKC-epsilon/DN blunted the acute increase in ERK1/2 phorphorylation induced by the alpha(1)-adrenergic agonist phenylephrine (PE ). Inhibition of PKC-delta with rottlerin potentiated the effects of PE on ERK1/2 phosphorylation. PKC-epsilon/DN adenovirus also blocked cardiocyte growth as measured after 48 h of PE treatment, although the multiplicity of infection was lower than that required to block acute ERK1/2 activation. PE activated p38 mitogen-activated protein kinase as measured by its phosphorylation, but the response was not blocked by PKC inhibitors or by overexpression of PKC-epsilon/DN. Taken together, these studies show that the hypertrophic agonist PE regulates ERK1/2 activity in cardiocytes by a pathway dependent on PKC-epsilon and that PE-induced growth is mediated by PKC-epsilon.     

Expression of protein kinase C isoenzymes in thymocyte subpopulations and their differential regulation

The expression of the different protein kinase C (PKC) isozymes in mouse thymocytes was studied to determine if there is a correlation between isozyme expression and thymocyte phenotype. Expression of PKC isozymes in thymocyte subsets (distinguished by the CD4 or CD8 Ag) was determined by message amplification phenotyping. The expression of mRNA for PKC-alpha, -beta, -epsilon, and -zeta, but not -gamma or -delta isozymes, was detected in all of the unstimulated thymocyte subpopulations analyzed. Thus no differences in the pattern of PKC isozyme expression were found that could be correlated with thymocyte phenotype. However, it was noted that the levels of PKC mRNA expression were affected by different stimuli in unfractionated thymocytes. Whereas mRNA levels of PKC-alpha and -beta were down-regulated by PMA and ionomycin treatment, no significant changes were seen in the levels of PKC-epsilon mRNA with these agents. PKC-epsilon mRNA decreased in thymocytes exposed to Con A similar to what has been reported for PKC-epsilon protein. PKC-zeta mRNA was also down-regulated by PMA or ionomycin, and the combination of both compounds caused a more rapid and drastic effect. Finally, PKC-delta mRNA expression was induced transiently in thymocytes only after exposure to PMA or Con A, and this induction was inhibited by ionomycin treatment. These results indicate that message levels of specific isoforms of PKC are uniquely regulated and suggest an additional level of control of PKC activity in activated lymphocytes.     

Role of BMP Signaling for the Formation of Auditory Brainstem Nuclei and Large Auditory Relay Synapses

Large excitatory synapses are found at specific points in the neuronal circuits of the auditory brainstem, to enable fast information transfer and the preservation of acoustic timing information. The extracellular cues and signaling mechanisms that lead to the development of these specialized synaptic connections, exemplified by the calyx of Held in the medial nucleus of the trapezoid body (MNTB), are still largely unknown. Here, we investigate the role of BMP signaling for the early development of the ventral cochlear nucleus (VCN) and MNTB, and for the initial formation of the calyx of Held synaptic connection. We used conditional alleles of two BMP type‐1 receptors in the background of a constitutive BMPR1b knock‐out (KO), or else a conditional allele of SMAD4. The conditional alleles were recombined by the Krox20^Cre mouse line that is active around mid‐gestation in rhombomeres (r) 3 and 5 from which the VCN and MNTB are derived; alternatively, virus‐mediated Cre‐expression was performed early postnatally in the VCN. The data show that embryonic SMAD‐dependent BMP‐signaling in r3 and r5 contributes to the histogenesis of auditory brainstem nuclei. On the other hand, BMP‐receptor signaling early postnatally in presynaptic neurons of the calyx of Held projection is necessary for correct axon branch retraction, which suggests a cell‐autonomous role of presynaptic BMP‐receptors in synapse elimination at the developing calyx of Held. Thus, our work dissects developmentally early and late roles of BMP‐signaling for the formation of auditory brainstem nuclei, and the highly specialized synaptic connectivity in these structures.     

The V5 domain of protein kinase C plays a critical role in determining the isoform-specific localization, translocation, and biological function of protein kinase C-delta and -epsilon

The catalytic domain of overexpressed protein kinase C (PKC)-delta mediates phorbol 12-myristate 13-acetate (PMA)-induced differentiation or apoptosis in appropriate model cell lines. To define the portions of the catalytic domain that are critical for these isozyme-specific functions, we constructed reciprocal chimeras, PKC-delta/epsilonV5 and -epsilon/deltaV5, by swapping the V5 domains of PKC-delta and -epsilon. PKC-delta/epsilonV5 failed to mediate PMA-induced differentiation of 32D cells, showing the essential nature of the V5 domain for PKC-delta's functionality. The other chimera, PKC-epsilon/deltaV5, endowed inactive PKC-epsilon with nearly all PKC-delta's apoptotic ability, confirming the importance of PKC-delta in this function. Green fluorescent protein (GFP)-tagged PKC-deltaV5 and -epsilon/deltaV5 in A7r5 cells showed substantial basal nuclear localization, while GFP-tagged PKC-epsilon and -delta/epsilonV5 showed significantly less, indicating that the V5 region of PKC-delta contains determinants critical to its nuclear distribution. PKC-epsilon/deltaV5-GFP showed much slower kinetics of translocation to membranes in response to PMA than parental PKC-epsilon, implicating the PKC-epsilonV5 domain in membrane targeting. Thus, the V5 domain is critical in several of the isozyme-specific functions of PKC-delta and -epsilon.     

Decreased protein kinase C-epsilon expression in hypertrophied cardiac ventricles induced by triiodothyronine treatment in the rat

To examine the effect of thyroid hormone-induced cardiac hypertrophy on PKC expression, changes in the expression of PKC isoforms were studied in hypertrophied cardiac ventricles induced by triiodothyronine (T3) injection in the rat. Injection with T3 for 8 days induced 49% increase in cardiac weight compared to controls. Immunoblot analysis of cardiac ventricular extracts showed the expression of PKC-delta, -epsilon, and -zeta in both control and T3-treated groups. The expression of PKC-epsilon decreased by 40% in hyperthyroid rat cardiac ventricles, while PKC-delta and -zeta expressions were barely affected. PKC-epsilon immunoreactivity decreased in both cytosol and membrane fractions. On the contrary, PKC-epsilon expression did not decrease in the extract of hypertrophied cardiac ventricles produced by aortic banding or aortocaval shunt. These results indicate that thyroid hormone down regulates PKC-epsilon expression in the hyperthyroid-mediated cardiac hypertrophy.     

Expression and characterization of protein kinase C-delta

A cDNA encoding protein kinase C-delta (PKC-delta) was isolated from a rat brain library. The coding region was subcloned into the expression vector pmt2 and transfected into COS-1 cells. Expression of the protein led to an 11-fold increase in activity as determined with a synthetic peptide based on the PKC-delta pseudosubstrate site. The Mr of PKC-delta as determined by SDS/PAGE and immunoblot analysis using anti-(PKC-delta C-terminal) antibodies was 77,000. The enzyme was purified to near homogeneity and showed total dependency on phospholipid and diacylglycerol (or phorbol esters) for activity. Like PKC-epsilon, PKC-delta displays no Ca2+ dependence for activation. The substrate specificity of PCK-delta is similar to that of PKC-epsilon but quite different from other PKCs.     

Role for PKC-epsilon in neuronal death induced by oxidative stress

We investigated which isoforms of PKCs can be modulated and what their roles are during l-buthionine-S,R-sulfoximine (BSO)-induced neuronal death. We observed the isoform specific translocation of PKC-epsilon from the soluble fraction to the particulate in cortical neurons treated with 10 mM BSO. The translocation of PKC-epsilon by BSO was blocked by antioxidant trolox, suggesting the PKC-epsilon as a downstream of reactive oxygen species (ROS) elevated by BSO. Trolox inhibited the ROS elevation and the neuronal death in BSO-treated cortical cells. The BSO-induced neuronal death was remarkably inhibited by both the pharmacological inhibition of PKC-epsilon with epsilonV1-2 and the functional blockade for PKC-epsilon through overexpression of PKC-epsilon V1 region, suggesting the detrimental role of PKC-epsilon. These results suggest that PKC-epsilon is the major PKC isoform involved in the pathways triggered by ROS, leading to neuronal death in BSO-treated cortical neurons.     

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.     

Role of PKC in autocrine regulation of rat ventricular K+ currents by angiotensin and endothelin

Transient and sustained K(+) currents were measured in isolated rat ventricular myocytes obtained from control, steptozotocin-induced (Type 1) diabetic, and hypothyroid rats. Both currents, attenuated by the endocrine abnormalities, were significantly augmented by in vitro incubation (>6 h) with the angiotensin-converting enzyme inhibitor quinapril or the angiotensin II (ANG II) receptor blocker saralasin. Western blots indicated a parallel increase in Kv4.2 and Kv1.2, channel proteins that underlie the transient and (part of the) sustained currents. Under diabetic and hypothyroid conditions, both currents were also augmented by an endothelin receptor blocker (PD142893) or by an endothelin-converting enzyme inhibitor. Kv4.2 density was also enhanced by PD142893. Incubation (>5 h) with the PKC inhibitor bis-indolylmaleimide augmented both currents, whereas the PKC activator dioctanoyl-rac-glycerol (DiC8) prevented the augmentation of currents by quinapril. DiC8 also prevented the augmentation of Kv4.2 density by quinapril. Specific peptides that activate PKC translocation indicated that PKC-epsilon and not PKC-delta is involved in ANG II action on these currents. In control myocytes, quinapril and PD142893 augmented the sustained late current but had no effect on peak current. It is concluded that an autocrine release of angiotensin and endothelin in diabetic and hypothyroid conditions attenuates K(+) currents by suppressing the synthesis of some K(+) channel proteins, with the effects mediated at least partially by PKC-epsilon.