Effects Of Protein Kinase C Inhibitors Research Articles

Potential Antiviral Target for SARS-CoV-2: A Key Early Responsive Kinase during Viral Entry

Currently, there is no effective antiviral medication for coronavirus disease 2019 (COVID-19) and the knowledge on the potential therapeutic target is in great need. Guided by a time-course transmi...

Inhibition of Sodium Glucose Cotransporter 2 Attenuates the Dysregulation of Kelch-Like 3 and NaCl Cotransporter in Obese Diabetic Mice.

Mechanisms underlying the frequent association between salt-sensitive hypertension and type 2 diabetes remain obscure. We previously found that protein kinase C (PKC) activation phosphorylates Kelch-like 3 (KLHL3), an E3 ubiquitin ligase component, at serine 433. We investigated whether impaired KLHL3 activity results in increased renal salt reabsorption via NaCl cotransporter (NCC). We used the db/db diabetes mouse model to explore KLHL3's role in renal salt handling in type 2 diabetes and evaluated mechanisms of KLHL3 dysregulation in cultured cells. We observed PKC activity in the db/db mouse kidney and phosphorylation of serine 433 in KLHL3 (KLHL3S433-P). This modification prevents binding of with-no-lysine (WNK) kinases; however, total KLHL3 levels were decreased, indicating severely impaired KLHL3 activity. This resulted in WNK accumulation, activating NCC in distal convoluted tubules. Ipragliflozin, a sodium glucose cotransporter 2 (SGLT2) inhibitor, lowered PKC activity in distal convoluted tubule cells and reduced KLHL3S433-P and NCC levels, whereas the thiazolidinedione pioglitazone did not, although the two agents similarly reduced in blood glucose levels. We found that, in human embryonic kidney cells expressing KLHL3 and distal convoluted tubule cells, cellular glucose accumulation increased KLHL3S433-P levels through PKC. Finally, the effect of PKC inhibition in the kidney of db/db mice confirmed PKC's causal role in KLHL3S433-P and NCC induction. Dysregulation of KLHL3 is involved in the pathophysiology of type 2 diabetes. These data offer a rationale for use of thiazide in individuals with diabetes and provide insights into the mechanism for cardiorenal protective effects of SGLT2 inhibitors.

Effect of PKC inhibition on cell migration and invasion of oral squamous cell carcinoma

By using HN4 cells, the correlation among protein kinase C(PKC), P120-catenin(P120ctn) and E-cadherin (E-cad) was investigated, and the role of them in migration and invasion of oral squamous-cell cancer(OSCC) was evaluated. Plasmid pGFP-V-RS-P120ctn shRNA was used to transfect HN4 cells to significantly reduce the expression of P120ctn and PKC inhibitor staurosporine (STS) was added. mRNA and protein expression of PKC, P120ctn and E-cad were tested and Transwell cell invasion and cell migration assay was used to test the invasion and migration capacity before and after PKC inhibition. Statistical analysis was performed using SPSS 18.0 software package. When PKC was inhibited by STS, the expression of P120ctn and E-cad in OSCC cells were increased and the migration and invasion capacity of tumor cells decreased significantly (P<0.05). CONCLUSIONS：PKC may be involved in the process of HN4 migration and invasion by regulating cell adhesion through the effect of P120ctn and E-cad.

Effect of Tamoxifen and Lithium on Treatment of Acute Mania Symptoms in Children and Adolescents.

Many studies have supported the role of protein kinase C (PKC) inhibitors in the physiopathology and treatment of bipolar disorder in adults. Tamoxifen is one of the drugs with the effect of PKC inhibition. This study aimed to determine the effect of tamoxifen on the rate of improvement mania symptoms in the sample of children and adolescents with acute mania. In this randomized, placebo-controlled clinical trial study, registered in www.irct.ir with the code of IRCT201410126418N3, overall 44 patients with bipolar disorder with acute manic episode were randomly assigned into treatment and control groups. The serum levels of lithium and tamoxifen among the participants in the treatment groups were 0.8 -1.1 mg and 20-40 mg per day respectively. Serum level of lithium among participants in the control group was similar. The main comparisons were made based on the Young Mania Rating Scale (YMRS) and Children Depression Inventory (CDI) scores of the participants at baseline and at the end of each study week. The pharmacological side effects of serum level of lithium were examined weekly. Analysis of Covariance(ANCOVA) test was used for the statistical analysis. There was no difference in the baseline score of YMRS and CDI in the treatment and control groups while a statistical significant difference (P < 0.05) in these scores was found between and within the groups. The addition of tamoxifen to lithium causes a significant difference in reducing the symptoms of mania and depression in the treatment group compared to the control group.

Madecassoside suppresses proliferation and invasiveness of HGF-induced human hepatocellular carcinoma cells via PKC-cMET-ERK1/2-COX-2-PGE2 pathway

Recent studies showed that Madecassoside (MAD), a pentacyclic triterpene isolated from Centella asitica (L.), was used as a therapeutic agent in wound healing and also as an anti-inflammatory, anti-oxidative activities and anti-aging agent. However, its role in cancer has not been elucidated. In our present study, hepatocyte growth factor (HGF) induced the phosphorylation of its corresponding receptor cMET, increased expression of cyclo-oxygenase-2 (COX-2) and prostaglandin E2 (PGE2) in human hepatocellular carcinoma (HCC) cells lines (HepG2 and SMMC-77), and this effect was inhibited by MAD in a dose-dependent manner. In addition, MAD exhibited significant anti-proliferative and anti-invasive effect in HGF-induced HepG2 and SMMC-77 cells. Moreover, MAD inhibited the phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and the protein kinase C (PKC) activity in HGF-induced HepG2 and SMMC-77 cells. This conclusion was consistent with the effect of selective COX-2 inhibitor (NS-398) and knockdown of COX-2 by siRNA on attenuating the proliferation and invasiveness potential, and over-expression of COX-2 on abolishing the effects of MAD on proliferation and invasiveness potential, and was also in parallel with the effect of PKC inhibitor (Bisindolylmaleimide) on inhibiting PKC activity, MEK/ERK1/2 inhibitor (PD98059) inhibited MEK/ERK1/2 pathways in HGF-induced HepG2 and SMMC-77 cells. Collectively, MAD could inhibit the HGF-activated proliferation and invasiveness of HCC cells via regulating the activation of cMET-PKC-ERK1/2-COX-2-PGE2 cascade, which indicated that MAD might help control HGF-linked HCC.

High-density lipoprotein of patients with breast cancer complicated with type 2 diabetes mellitus promotes cancer cells adhesion to vascular endothelium via ICAM-1 and VCAM-1 upregulation.

Adhesion of disseminating tumor cells to vascular endothelium is a pivotal starting point in the metastasis cascade. We have shown previously that diabetic high-density lipoprotein (HDL) has the capability of promoting breast cancer metastasis, and this report summarizes our more recent work studying the role of abnormal HDL in facilitating the adhesion of the circulating tumor cells to the endothelium. This is an initiating step in breast cancer metastasis, and this work assesses the role of ICAM-1 and VCAM-1 in this process. MDA-MB-231, MCF 7, and human umbilical vein endothelial cells (HUVECs) were treated with normal HDL from healthy controls (N-HDL), HDL from breast cancer patients (B-HDL), or HDL from breast cancer patients complicated with type 2 diabetes mellitus (BD-HDL), and the cell adhesion abilities were determined. ICAM-1 and VCAM-1 expression as well as the protein kinase C (PKC) activity were evaluated. The effect of PKC inhibitor and PKC siRNA on adhesion was also studied. The immunohistochemical staining of ICAM-1, VCAM-1, and E-selectin from breast cancer patients and breast cancer patients complicated with type 2 diabetes mellitus (T2DM) were examined. Our results indicate that BD-HDL promoted an increase in breast cancer cell adhesion to HUVECs and stimulated higher ICAM-1 and VCAM-1 expression on the cells surface of both breast cancer and HUVEC cells, along with the activation of PKC. Increased tumor cell (TC)-HUVEC adhesion, as well as ICAM-1 and VCAM-1 expression induced by BD-HDL, could be inhibited by staurosporine and PKC siRNA. In addition, a Db/db type 2 diabetes mouse model has more TC-Vascular Endothelium adhesion compared to a normal model. However, BD patients have a lower expression of ICAM-1, VCAM-1, and E-selectin in their tumor tissues. BD-HDL facilitates the adhesion of tumor cells to vascular endothelium by upregulating the expression of ICAM-1 and VCAM-1, thereby promoting the initial progression of breast cancer metastasis. This work indicates a prospective utilization of HDL-based strategies in the treatment of breast cancer patients with type 2 diabetes.

Nanog expression is negatively regulated by protein kinase C activities in human cancer cell lines

Nanog is a transcription factor that is essential for the maintenance of pluripotency of the embryonic stem cells. Nanog has been shown to be expressed in various kinds of human tumors, suggesting a role in tumorigenesis. In this study, we found that Nanog expression was upregulated by inhibition of protein kinase C (PKC) activity in six human cancer cell lines examined. In a Nanog non-expressing human nasopharyngeal carcinoma cell line, NPC-076, Nanog mRNA level and protein level were both induced and dose-dependently promoted by exposure to PKC inhibitors. Knockdown experiments showed that PKCα and PKCδ were two subtypes exerted most of the effect. The reporter assay showed that Nanog promoter activity was promoted by exposure of the cells to PKC inhibitors and the effect was dependent on the presence of the Octamer-Sox composite element. The involvement of Octamer-Sox composite element was further supported by the observation that silencing of Oct4 and Sox2 in NPC-076 cells attenuated the effects of PKC inhibitors. In Nanog-expressing human embryonal carcinoma cell lines, NT2/D1 and NCCIT, Nanog expression was suppressed by exposure to PKC activator Phorbol-12-myristate-13-acetate (PMA). Further study showed that overexpression of PKCα elicited a repressive effect on Nanog expression in NT2/D1 cells. Consistently, mutation of the Octamer-Sox composite element abolished the suppressive effect by PKC activator. Nanog expression was of cellular significance in that ectopic expression in NPC-076 stimulated cell proliferation and knockdown of the endogenous Nanog expression in NT2/D1-suppressed cell proliferation.

Task demands dissociate the effects of muscarinic M1 receptor blockade and protein kinase C inhibition on attentional performance in rats

The cholinergic system is known to be necessary for normal attentional processing. However, the receptors and mechanisms mediating the effects of acetylcholine on attention remain unclear. Previous work in our laboratory suggested that cholinergic muscarinic receptors are critical for maintaining performance in an attention-demanding task in rats. We examined the role of the muscarinic M(1) receptor and protein kinase C (PKC), which is activated by the M(1) receptor, in attention task performance. Rats were trained in an attention-demanding task requiring discrimination of brief (500, 100, 25 ms) visual signals from trials with no signal presentation. The effects of muscarinic M(1) receptor blockade were assessed by administering dicyclomine (0-5.0 mg/kg). The effects of PKC inhibition were assessed by administering chelerythrine chloride (0-2.0 mg/kg). Dicyclomine decreased the accuracy of detecting longer signals in this attention task, including when attentional demands were increased by flashing a houselight throughout the session. Chelerythrine chloride decreased the accuracy of signal detection in the standard version of the task but not when the houselight was flashed throughout the session. The present findings indicate that muscarinic M(1) receptors are critical for maintaining performance when attentional demands are increased, and that PKC activity may contribute to some aspects of attentional performance.

Vascular protective effects of selective PKCb inhibitor with Ruboxistaurin in Murine models

Shi-Fang Yan New York University School of Medicine, USA A a growing body of evidence from animal and human studies indicates benefi cial eff ects of protein kinase C  (PKC) inhibition on microvascular parameters, our studies have examined the eff ects of PKC inhibitor, ruboxistaurin, on macrovascular disease in the experimental murine models of atherosclerosis, restenosis, myocardial ischemia, and lung ischemia. Wild type C57BL6 and apoE-/male mice were maintained on normal rodent chow and then fed normal chow containing the PKCβ inhibitor, ruboxistaurin (LY333531) or vehicle chow without inhibitor. Mice were rendered diabetic (D) (defi ned as plasma glucose >250 mg/ dl) by injection of streptozotocin (65μg/g weight) for fi ve consecutive days at age 6 weeks. Our data provide the fi rst evidence that key species and stresses implicated in vascular injury, such as modifi ed lipoproteins, glucose and diacylglycerol (DAG), acute physical stress, hypoxia and ischemia/reperfusion transduce their key pathogenic eff ects, at least in part, via rapid and, in certain settings, chronic recruitment of PKC signaling pathway. Th is work raises the possibility that blockade of the PKC by a pharmacologic inhibitor, ruboxistaurin, may attenuate neointimal expansion or organ dysfunction and damage triggered by acute mechanical injury, chronic atherosclerosis, or ischemia-reperfusion stress. In addition, based on the bioavailability and tolerability of ruboxistaurin in diabetes and our fi ndings in euglycemia and diabetic atherosclerosis, we speculate that blockade of PKC signaling pathway may hold promise as a therapeutic intervention in treating macrovascular disease involving the heart and large vessels in both diabetes and non-diabetes, although this remains to be proven in clinical trials.

PKC inhibition increases gap junction intercellular communication and cell adhesion in human neuroblastoma

Gap junction intercellular communication and cell-cell adhesion are essential for maintaining a normal cellular phenotype, including the control of growth and proliferation. Loss of either cell-cell adhesion or communication is common in cancers, while restoration of function is associated with tumor suppression. Protein kinase C (PKC) isozymes regulate a broad spectrum of cellular functions including growth and proliferation, and their overexpression has been correlated with carcinogenesis. Consequently, PKC inhibitors are currently undergoing clinical trials as an anti-cancer agents although the precise cellular alterations induced by PKC inhibitors remain to be elucidated. In the current study, the effects of PKC inhibitors on cell interactions were investigated using human neuroblastoma (IMR32, SKNMC, and SHSY-5Y) cell lines. An analysis of intercellular communication revealed an increase in gap junctional coupling with PKC inhibition. The observed increase in coupling was not associated with a change in Connexin 43 distribution or an alteration of phosphorylation status of the protein. There was also an increase in cell-cell adhesion with PKC inhibitor treatment as indicated by a cell aggregation assay. Therefore, the growth suppressive abilities of PKC inhibition on tumors may be due to the cancer suppressive effects of increased gap junction intercellular communication and cell-cell adhesion.

Some different effect of PKC inhibitors on the acetylcholine, and endothelin-1-induced contractions of rat bronchial smooth muscle

Although the role of protein kinase C (PKC) has been suggested in agonist-induced bronchial smooth muscle contractions, the PKC isoform(s) involved in the phenomenon are not yet clear now. Moreover, contractile agonists may have different signaling pathways in airway smooth muscle contraction. Therefore, the identification of PKC isoforms playing a role in rat bronchial smooth muscle was investigated. The presence of PKCα, β, γ, δ, ε, η, ζ, λ and ι isoforms was determined by immunoblot in the muscle. The effects of four PKC inhibitors, GF109203X, calphostin C, Gö6976 and rottlerin on acetylcholine and endothelin-1-induced bronchial smooth muscle contractions were then examined. The inhibitors were cumulatively administered after the acetylcholine or endothelin-1-induced contraction reached plateau. Since the acetylcholine-induced bronchial smooth muscle contraction was significantly inhibited by GF109203X (PKCs α, β, γ, δ and ε inhibitor) and calphostin C (conventional and novel PKCs inhibitor) but not by Gö6976 (PKCs α, β and γ inhibitor) and rottlerin (PKCs δ and θ inhibitor), novel PKCs might be involved in the acetylcholine-induced contraction. On the other hand, the bronchial smooth muscle contraction by endothelin-1 was significantly inhibited by GF109203X, calphostin C and Gö6976 but not by rottlerin. The conventional PKC might partially play important role in the endothelin-1 induced contraction. In conclusion, the present study clearly demonstrated the expression of multiple PKC isoforms in rat bronchial smooth muscle. Some different PKC isoforms might be partly involved between the acetylcholine and endothelin-1-induced bronchial smooth muscle contractions in rats.

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 (α, 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.

Probable involvement of epsilon-isoform of protein kinase C in rat bronchial smooth muscle contraction induced by acetylcholine

Although the role of protein kinase C (PKC) has been suggested in agonist-induced bronchial smooth muscle contraction, the PKC isoform(s) involved in this phenomenon is not clear now. In the present study, the effects of three PKC inhibitors, GF1092603X, Gö6976 and rottlerin on acetylcholine (ACh)-induced bronchial smooth muscle contraction were examined to identify the PKC isoform(s) involved in the contraction. Bronchial smooth muscles were pretreated with each PKC inhibitor (10(-6) and 10(-5) M) 30 min before cumulative administration of ACh. In another series of experiments, the effects of PKC inhibitors on the maximal contraction induced by 10(-3) M ACh were determined: the inhibitors were cumulatively administered (10(-8)- 10(-5) M) after the ACh-induced contraction reached plateau. The ACh-induced bronchial smooth muscle contraction was significantly inhibited by GF109203X (inhibitor of PKC alpha, beta, gamma, delta and epsilon) but not by Gö6976 (PKC delta, beta and gamma inhibitor) and rottlerin (PKC delta and theta inhibitor). Moreover, mRNA and protein of PKC epsilon were detected in rat bronchial smooth muscle. Taken together, PKC epsilon might be involved in the ACh-induced bronchial smooth muscle contraction in rats.

Interdependence of PKC-Dependent and PKC-Independent Pathways for Presynaptic Plasticity

Diacylglycerol (DAG) is a prominent endogenous modulator of synaptic transmission. Recent studies proposed two apparently incompatible pathways, via protein kinase C (PKC) and via Munc13. Here we show how these two pathways converge. First, we confirm that DAG analogs indeed continue to potentiate transmission after PKC inhibition (the Munc13 pathway), but only in neurons that previously experienced DAG analogs, before PKC inhibition started. Second, we identify an essential PKC pathway by expressing a PKC-insensitive Munc18-1 mutant in munc18-1 null mutant neurons. This mutant supported basic transmission, but not DAG-induced potentiation and vesicle redistribution. Moreover, synaptic depression was increased, but not Ca2+-independent release evoked by hypertonic solutions. These data show that activation of both PKC-dependent and -independent pathways (via Munc13) are required for DAG-induced potentiation. Munc18-1 is an essential downstream target in the PKC pathway. This pathway is of general importance for presynaptic plasticity.

HIGH GLUCOSE‐INDUCED HUMAN UMBILICAL VEIN ENDOTHELIAL CELL HYPERPERMEABILITY IS DEPENDENT ON PROTEIN KINASE C ACTIVATION AND INDEPENDENT OF THE Ca2+–NITRIC OXIDE SIGNALLING PATHWAY

1. Endothelial barrier dysfunction plays a pivotal role in the pathogenesis of diabetic vascular complications. The precise molecular mechanisms by which hyperglycaemia causes the increased permeability in endothelial cells are not yet well understood. In the present study, we investigated whether high concentrations of glucose induce endothelial permeability through the activation of protein kinase C (PKC) and/or the calcium-nitric oxide (NO) signalling pathway in human umbilical vein endothelial cells (HUVEC). 2. Endothelial permeability was measured by albumin diffusion across endothelial monolayers under the stimuli of high glucose (HG; 20 mmol/L), 100 nmol/L phorbol-myristate-acetate (PMA) or 100 nmol/L histamine. The intracellular calcium concentration ([Ca2+]i) was detected in HUVEC using the fluorescent probe fura-2 AM. The effects of PKC inhibitors (LY379196 and hypocrellin A) and the NO synthase (NOS) inhibitor NG-monomethyl-L-arginine (L-NMMA) on endothelial permeability and [Ca2+]i were determined. 3. High glucose and PMA increased endothelial permeability associated with decreased [Ca2+]i, whereas histamine triggered significant increases in endothelial permeability, accompanied by increases in [Ca2+]i in HUVEC. Hypocrellin A (HA) and LY379196 reversed both HG- and histamine-induced endothelial permeability. The NOS inhibitor L-NMMA only abolished histamine- and not HG-induced endothelial permeability. Neither LY379196, HA nor L-NMMA had any significant effects on alterations in [Ca2+]i caused by HG and histamine. 4. These results indicate that increased endothelial permeability in HUVEC induced by HG is dependent on PKC activity and is independent of the [Ca2+]i-NO pathway. Increased endothelial permeability due to other inflammatory factors, such as histamine, may also be mediated by the PKC pathway. Thus, PKC inhibitors would be a potential therapeutic approach to endothelial dysfunction induced by hyperglycaemia, as well as other inflammatory factors, in diabetes.

Regulation of hyaluronan secretion into rabbit synovial joints in vivo by protein kinase C.

Hyaluronan (HA) is important for joint cavitation, lubrication, volume regulation and synovial fluid drainage but little is known about the regulation of joint HA synthesis/secretion in vivo. We investigated whether HA secretion into joints in vivo can be regulated by protein kinase C (PKC). Secretion into the knee joint cavity of anaesthetised rabbits was measured over 6 h by washout and chromatography. Joints received intra-articular injections of Ringer vehicle (control) or an activator of classical PKC isoforms, phorbol-12-myristate-13-acetate (PMA), at 20-2000 ng ml(-1). The effects of PKC inhibition by bisindolylmaleimide (BIM) and protein synthesis inhibition by cycloheximide (CX) on basal and stimulated HA secretion were also studied. The endogenous HA mass, 181+/-8 microg (n=26, mean +/- S.E.M.), and basal secretion rate, 4.4+/-0.4 microg h(-1), indicated a turnover time of 41 h. Secretion rate showed a dose-dependent response to PMA (n=30), rising 5-fold to 21.7+/-5.0 microg h(-1) (n=5) at 2000 ng ml(-1) PMA (P<0.0001, one-way ANOVA). PMA-induced stimulation was partially suppressed by CX (HA secretion: 5.8+/-1.7 microg h(-1), n=8, P<0.01) and totally blocked by BIM (HA secretion: 3.2+/-0.6 microg h(-1), n=9, P<0.001). Basal HA secretion was unaffected by CX over 6 h (4.2+/-0.7 microg h(-1), n=8) but was reduced by 29 % by BIM (3.1+/-0.6 microg h(-1), n=10, P=0.03). It is concluded that: (1) PKC can stimulate HA secretion into joints in vivo through mechanisms involving protein synthesis de novo as well as phosphorylation; (2) basal HA secretion is only partially PKC dependent; and (3) hyaluronan synthase turnover time is >6 h in vivo, which is slower than in vitro (<2-3 h).

Protein kinase C inhibits the transplasma membrane influx of Ca2+ triggered by 4-aminopyridine in Jurkat T lymphocytes.

4-aminopyridine (4AP) is a general blocker of voltage-dependent K+ channels. This pyridine derivative has also been shown to inhibit T cell proliferation, to modulate immune responses and to alleviate some of the symptoms associated with neurological disorders such as multiple sclerosis, myasthenia gravis and Alzheimer's disease. 4AP triggers a Ca2+ response in lymphocytes, astrocytes, neurons and muscle cells but little is known about the regulation of the 4AP response in these cells. We report that 4AP induced a non-capacitative transplasma membrane influx of Ca2+ in Jurkat T lymphocytes. The influx of Ca2+ was not affected by activation or inhibition of protein kinase A (PKA). In contrast, activation of protein kinase C (PKC) by phorbol myristyl acetate (PMA), mezerein or 1-oleoyl-2-acetyl-sn-glycerol (OAG) inhibited the influx of Ca2+ triggered by 4AP. The inhibitory effect of PKC could be prevented by prior exposure of the cells to the PKC inhibitor GF 109203X. Under these conditions, mezerein and OAG no longer inhibited the 4AP-dependent Ca2+ response. Inhibition of serine and threonine protein phosphatases PP1 and PP2A by treating the cells with calyculin A (CalA) reduced the Ca2+ response to 4AP. Okadaic acid (OA) had no effect, suggesting an involvement of PP1. A combination of CalA and OAG (or PMA) abolished the influx of Ca2+ induced by 4AP, adding further evidence to the importance of protein phosphorylation in the modulation of the 4AP response. Our data suggest that the transplasma membrane influx of Ca2+ triggered by 4AP in Jurkat T cells can be modulated by the opposite actions of PKC and protein serine and threonine phosphatase(s).

Dual regulation of the parathyroid hormone (PTH)/PTH-related peptide receptor signaling by protein kinase C and beta-arrestins.

We examined here the role of second messenger-dependent kinases and beta-arrestins in short-term regulation of the PTH receptor (PTHR) signaling. The inhibition of protein kinase C (PKC) in COS-7 cells transiently expressing PTHR, led to an approximately 2-fold increase in PTH-stimulated inositol phosphate (IP) and cAMP production. The inhibition of protein kinase A increased cAMP production 1.5-fold without affecting IP signaling. The effects of PKC inhibition on PTHR-mediated G(q) signaling were strongly decreased for a carboxy-terminally truncated PTHR (T480) that is phosphorylation deficient. PKC inhibition was associated with a decrease in agonist-stimulated PTHR phosphorylation and internalization without blocking PTH-dependent mobilization of beta-arrestin2 to the plasma membrane. Overexpression of beta-arrestins strongly decreased the PTHR-mediated IP signal, whereas cAMP production was impaired to a much lower extent. The regulation of PTH-stimulated signals by beta-arrestins was impaired for the truncated T480 receptor. Our data reveal mechanisms at, and distal to, the receptor regulating PTHR-mediated signaling pathways by second messenger-dependent kinases. We conclude that regulation of PTHR-mediated signaling by PKC and beta-arrestins are separable phenomena that both involve the carboxy terminus of the receptor. A major role for PKC and beta-arrestins in preferential regulation of PTHR-mediated G(q) signaling by independent mechanisms at the receptor level was established.

Inhibitory effects of protein kinase C on inwardly rectifying K+- and ATP-sensitive K+ channel-mediated responses of the basilar artery.

The structurally related, inwardly rectifying K+ (K(IR)) channel and the ATP-sensitive K+ (K(ATP)) channel are important modulators of cerebral artery tone. Although protein kinase C (PKC) activators have been shown to inhibit these channels with the use of patch-clamp electrophysiology, effects of PKC on K+ channel function in intact cerebral blood vessels are unknown. We therefore tested whether pharmacological alteration of PKC activity affects cerebral vasodilator responses to K(IR) and/or K(ATP) channel activators in vivo. We measured changes in basilar artery diameter using a cranial window preparation in anesthetized rats. In addition, intracellular recordings of smooth muscle membrane potential were made in isolated basilar arteries. K+ (5 to 15 mmol/L) and aprikalim (1 to 10 micromol/L) each elicited reproducible vasodilatation. The PKC activator phorbol-12,13-dibutyrate (PdBu) (50 nmol/L) inhibited responses to K+ (by 40% to 55%) and aprikalim (by 40% to 70%), whereas responses to papaverine were unaffected. The PKC inhibitor calphostin C (0.1 micromol/L) augmented responses to K+ (by 2- to 3-fold) and aprikalim (2-fold) but not papaverine. In addition, K+ (5 mmol/L) and aprikalim (3 micromol/L) each hyperpolarized the basilar artery. PdBu inhibited these responses to aprikalim by 45% but had no effect on K+-induced hyperpolarization. These data suggest that both basal and stimulated PKC activity inhibit K(IR) and K(ATP) channel-mediated cerebral vasodilatation in vivo. The inhibitory effect on K(ATP) channel-mediated vasodilatation occurs at least partly by inhibition of hyperpolarization mediated by K(ATP) channels. PKC inhibits K+-induced vasodilatation without affecting hyperpolarization, suggesting that the inhibitory effect of PKC on vasodilator responses to K+ does not involve altered K(IR) channel function.

Differential effects of protein kinase C on human vascular smooth muscle cell proliferation and migration.

Vascular smooth muscle cell (SMC) migration and proliferation contribute to intimal hyperplasia, and protein kinase C (PKC) may be required for both events. In this report, we investigated the role of PKC in proliferation and migration of SMC derived from the human saphenous vein. Activation of PKC by phorbol-12,13-dibutyrate (PDBu) or (-)-indolactam [(-)-ILV] increases SMC proliferation. Downregulation of PKC activity by prolonged incubation with phorbol ester or inhibition of PKC with chelerythrine in SMC diminished agonist-stimulated proliferation. In contrast, stimulation of PKC with PDBu or (-)-ILV inhibited basal and agonist-induced SMC chemotaxis. Moreover, downregulation of PKC or inhibition with chelerythrine accentuated migration. We postulated that the inhibitory effect of PKC on SMC chemotaxis was mediated through cAMP-dependent protein kinase (protein kinase A, PKA). In support of this hypothesis, we found that activation of PKC in SMC stimulated PKA activity. The cAMP agonist forskolin significantly inhibited SMC chemotaxis. Furthermore, the inhibitory effect of PKC on SMC chemotaxis was completely reversed by cAMP or PKA inhibitors. In search of the PKC isotype(s) underlying these differential effects of PKC in SMC, we identified eight isotypes expressed in human SMC. Only PKC-alpha, -beta I, -delta, and -epsilon were eliminated by downregulation, suggesting that one or more of these four enzymes facilitate the observed phorbol ester-dependent effects of PKC in SMC. In summary, we found that PKC activation enhances proliferation but inhibits migration of human vascular SMC. These differential effect of PKC on vascular cells appears to be mediated through PKC-alpha, -beta I, -delta, and/or -epsilon.