Activation of keratinization of keratinocytes from fetal rat skin with N-(O-linoleoyl) omega-hydroxy fatty acyl sphingosyl glucose (lipokeratinogenoside) as a marker of epidermis

To elucidate the functional significance of sphingolipids altered in the epidermal differentiation, we examined, the effects of sphingolipids on the activity of transglutaminase and the formation of cornified envelopes in the keratinocytes from fetal rat skin. N-(O-linoleoyl) omega-hydroxy fatty acyl sphingosyl glucose (lipokeratinogenoside) that was characteristically contained in the mammalian epidermis, as well as nonhydroxy fatty acid-containing GalCer and GlcCer, significantly enhanced the activity and the formation, but no or rather inhibited activity was observed with ceramides, GalCer with alpha-hydroxy fatty acid, saponified lipokeratinogenoside, etc. This indicates that skin-characteristic lipokeratinogenoside functions to regulate the transglutaminase for the formation of cornified envelopes in the process of keratinization.     

Sphingosine and phorbol ester modulate protein kinase C activity and modify ATP-evoked calcium mobilization in glioma C6 cells.

The effect of sphingosine and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on ATP-evoked Ca(2+) mobilization in glioma C6 cells was studied with the Fura-2 video-imaging technique. Treatment of the cells with TPA, an activator of protein kinase C, reduced the ATP-evoked release of Ca(2+) from the intracellular stores, whereas sphingosine, known from in vitro studies as a protein kinase C inhibitor, potentiated Ca(2+) release synergistically with ATP. ATP-induced Ca(2+) mobilization was also enhanced by a specific protein kinase C inhibitor, GF 109203X. Pretreatment of the cells with GF 109203X prevented TPA action, whereas TPA diminished the stimulatory effect of sphingosine. However, this sphingosine effect was only observed after a short (1 min) treatment, whereas a longer treatment (5 min) reduced ATP-evoked Ca(2+) release. It is therefore concluded that sphingosine has two apparent actions: it inhibits protein kinase C providing a positive feedback regulation of receptor signals and it releases Ca(2+) from intracellular stores by an unknown mechanism, possibly independent of protein kinase C.     

The tumor promoter, 12-O-tetradecanoylphorbol-13-acetate accelerates keratinocyte differentiation and stimulates growth of an unidentified cell type in cultured human epidermis

Studies were conducted to determine the effects of the mouse skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on cultured human epidermal cells for comparison with known effects on mouse keratinocytes. In contrast to its effect on mouse cells, TPA did not stimulate human epidermal cell DNA synthesis. TPA stimulated differentiation in human keratinocytes resulting in sloughing of many cells by the 3rd day after exposure. Quantitative assays revealed that 50% of the TPA-exposed population was composed of cornified cells as opposed to 8% in untreated controls. A morphologically distinct cell type (TT cell) emerged after TPA treatment which was triangular in shape, did not stratify, appeared to proliferate rapidly and at most TPA concentrations became the predominant cell type within 1–2 weeks. Cultures composed predominantly of TT cells formed few cornified envelopes, grew well in the absence of TPA and formed colonies at low cell input. In contrast to its effect on keratinocytes, TPA enhanced TT colony formation 3–4-fold and decreased the doubling time of TT cells. Studies were performed to determine the origin of TT cells. Immunofluorescent staining indicated that TT cells lacked the keratinocyte antigens keratin, pemphigus and pemphigoid. Tonofilaments and desmosomes were not seen by electron microscopy. The lack of both melanosomes and standard histochemical DOPA oxidase staining indicated that TT cells were probably not of melanocyte origin. Tests used to identify Langerhans cells were negative. Whereas TT cells, as well as dermal fibroblasts, yielded positive immunofluorescence with antibodies to vimentin, TT cells gave a weak histochemical leucine aminopeptidase reaction, while the reaction of fibroblasts exposed to TPA was strong. Treatment of human dermal fibroblasts with TPA did not yield TT cells. The endothelial cell antigen factor VIII-associated protein was absent by immunofluorescence. These results suggest that the primary effect of TPA on cultured human epidermis is to accelerate terminal differentiation in the keratinocyte population and to stimulate growth of an as yet unidentified cell type.     

Potentiation of thyrotropin releasing hormone-induced inositol phospholipid metabolism and Ca2+ mobilization by cyclic AMP-increasing agents

Thyrotropin releasing hormone (TRH) caused significant breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) in GH3 cells, but vasoactive intestinal peptide (VIP) did not. However, VIP enhanced the TRH-induced hydrolysis of PIP2, the conversion of phosphatidylinositol 4-phosphate (PIP) to PIP2 and the accumulation of phosphatidic acid (PA). On the other hand, the tumor promoter, tetradecanoyl phorbol acetate (TPA), suppressed the TRH-induced hydrolysis of PIP2. In the membrane fraction, the addition of cAMP inhibited the PI kinase activity in a dose-dependent manner, but stimulated the PIP kinase activity. TPA did not affect the PI and PIP kinase activities at all. VIP enhanced the first spike phase of the TRH-induced increase in the intracellular Ca2+ level, while TPA inhibited such Ca2+ mobilization. These results suggested that cAMP-increasing agents enhanced inositol phospholipid metabolism and Ca2+ mobilization induced by TRH in GH3 cells but that TPA inhibited them.     

Characterization of sciellin, a precursor to the cornified envelope of human keratinocytes

The cornified envelope, located beneath the plasma membrane of terminally differentiated keratinocytes, is formed as protein precursors are cross-linked by a membrane associated transglutaminase. This report characterizes a new precursor to the cornified envelope. A monoclonal antibody derived from mice immunized with cornified envelopes of human cultured keratinocytes stained the periphery of more differentiated cells in epidermis and other stratified squamous epithelia including hair and nails. The epitope was widely conserved among mammals as determined by immunohistochemical and Western analysis. Immunoelectron microscopy localized the epitope to the cell periphery in the upper stratum spinosum and granulosum of epidermis. In the hair follicle, the epitope was present in the internal root sheath and in the infundibulum, the innermost aspect of the external root sheath. The antibody recognized a protein of relative mobility (M(r)) 82,000, pI 7.8. The protein was a transglutaminase substrate as shown by a dansylcadaverine incorporation assay. Purified cornified envelopes absorbed the reactivity of the antibody to the partially purified protein and cleavage of envelopes by cyanogen bromide resulted in release of immunoreactive fragments. The protein was soluble only in denaturing buffers such as 8 M urea or 2% sodium dodecyl-sulfate (SDS). Partial solubility could be achieved in 50 mM TRIS pH 8.3 plus 0.3 M NaCl (high salt buffer); the presence of a reducing agent did not affect solubility. Extraction of cultured keratinocytes in 8 M urea and subsequent dialysis against 50 mM TRIS pH 8.3 buffer resulted in precipitation of the protein with the keratin filaments. Dialysis against high salt buffer prevented precipitation of the protein. The unique solubility properties of this protein suggest that it aggregates with itself and/or with keratin filaments. The possible role of the protein in cornified envelope assembly is discussed. We have named this protein Sciellin (from the old english "sciell" for shell).     

Interaction of protein kinase C with filamentous actin: isozyme specificity resulting from divergent phorbol ester and calcium dependencies

The mechanism of activation of protein kinase C isoforms by filamentous actin (F-actin) was investigated with respect to isozyme specificity and phorbol ester and Ca(2+) dependencies. It was found that the "conventional" (cPKC), alpha, betaI, betaII, and gamma, "novel" (nPKC) delta and epsilon, and "atypical" (aPKC) zeta isoforms were each activated by F-actin with varying potencies. The level of activity along with the affinity for binding to F-actin was further potentiated by the phorbol ester 4beta-12-O-tetradecanoylphorbol 13-acetate (TPA), the potency of which again varied for each isoform. By contrast to the other cPKC isoforms, the level of cPKC-gamma activity was unaffected by TPA, as was also the case for aPKC-zeta. It was found that whereas in the absence of F-actin the soluble form of cPKC-betaI contained two phorbol ester binding sites of low and high affinity, respectively, as previously reported for cPKC-alpha [Slater et al. (1998) J. Biol. Chem. 273, 23160-23168], the F-actin-bound form of the isozyme contained only a single site of relatively low affinity. The level of TPA required to induce cPKC-alpha, -betaI, and -betaII activity and the binding of these isozymes to F-actin was reduced in the presence of Ca(2+). By contrast, the activity of cPKC-gamma was unaffected by Ca(2+), as were the activities of nPKC-delta and -epsilon and aPKC-zeta, as expected. Thus, the interaction with F-actin appears to be a general property of each of the seven PKC isozymes tested. However, isoform specificity may, in part, be directed by differences in the phorbol ester and Ca(2+) dependences, which, with the notable exception of cPKC-gamma, appear to resemble those observed for the activation of each isoform by membrane association. The observation that cPKC isoforms may translocate to F-actin as well as the membrane as a response to an elevation of Ca(2+) levels may allow for the functional coupling of fluctuations of intracellular Ca(2+) levels through cPKC to F-actin cytoskeleton-mediated processes.     

Ca(2+)-independent, phospholipid-activated protein kinase in 3Y1 cells.

We obtained a Ca(2+)-independent but 12-O-tetradecanoyl phorbol ester (TPA).phospholipid-activated protein kinase from rat embryo fibroblast 3Y1 cells by succeeding steps of DEAE-cellulose, H-9 affinity, and hydroxylapatite chromatography. This kinase was separated chromatography. This kinase was separated from a conventional PKC (Type III), by H-9 affinity column chromatography. The major peak from H-9 affinity column was eluted at 0.4 M of arginine and on the following step of hydroxylapatite column chromatography, at the KPO4 concentration of 0.1 M. The enzyme could be stimulated by phospholipids and by the tumor promoter TPA, but did not respond to calcium. The Ca(2+)-independent, phospholipid-activated protein kinase activity was susceptible to the protein kinase C inhibitors H-7 and K252a, but showed a phospholipid dependency and substrate specificity distinct from the conventional types of PKC. This protein kinase did not react with monoclonal antibodies against Types I, II, and III PKC. The activity of this enzyme was specifically reduced by immunoprecipitation, depending on the concentration of the polyclonal antibody, PC-delta, which was raised against a peptide synthesized according to a sequence of rat brain nPKC delta. The enzyme had a Mr of 76,000 as estimated by Western blotting. These results provide evidence for a unique type of Ca(2+)-independent, phospholipid-activated kinase, as expressed in 3Y1 cells.     

Phosphorylation of the inhibitory guanine-nucleotide-binding protein as a possible mechanism of inhibition by protein kinase C of agonist-induced Ca2+ mobilization in human platelet.

Increases in the intracellular Ca2+ concentration of human platelets caused by receptor agonists, such as thrombin, 9,11-epithio-11,12-methanothromboxane A2 (STA2), platelet-activating factor (PAF) and arginine-vasopressin, were inhibited by prior addition of 12-O-tetradecanoylphorbol 13-acetate (TPA) in time-dependent and concentration-dependent manners. The inhibitions were mostly reversed by staurosporine, and inhibitor of protein kinase C, added 1 min before TPA. Prior treatment of platelets with thrombin or STA2, the efficacious Ca2+ mobilizer, suppressed the increase in the intracellular Ca2+ concentration of the cells to other agonists, but treatment with less efficacious PAF or vasopressin did not. The heterologous receptor desensitizations were also reversed by staurosporine. The antibody, directed against the carboxy-terminal region of the alpha subunits 1 and 2 of the inhibitory guanine-nucleotide-binding proteins (Gi1 alpha and Gi2 alpha), was raised in rabbit and was used to immunoprecipitate Gi alpha in 32P-labeled platelets. The radioactivity was detected in Gi alpha after incubation of 32P-labeled platelets with TPA, thrombin or STA2, but not in the cells incubated with PAF or vasopressin. The time-dependency or concentration-dependency of TPA-induced phosphorylation of Gi alpha was similar to the dependency of its inhibitory action on agonist-induced Ca2+ mobilization. Thus, strong activation of Ca2+/phospholipid-dependent protein kinase C by phorbol ester or agonists of certain Ca(2+)-mobilizing receptors leads to phosphorylation of the alpha subunit of guanine-nucleotide-binding protein, thereby impairing the coupling of the G protein to receptors as a feedback regulatory component of the receptor-triggered intracellular Ca(2+)-mobilizing system.     

Protein kinase A and C are "Gatekeepers" of capacitative Ca2+ entry in the prothoracic gland cells of the silkworm, Bombyx mori

Application of protein kinases A and C inhibitors to the prothoracic glands cells of the silkworm, Bombyx mori, resulted in slow and gradual increases in intracellular Ca(2+) ([Ca(2+)](i)). Pharmacological manipulation of the Ca(2+) signalling cascades in the prothoracic gland cells of B. mori suggests that these increases of [Ca(2+)](i) are mediated neither by voltage-gated Ca(2+) channels nor by intracellular Ca(2+) stores. Rather they result from slow Ca(2+) leak from plasma membrane Ca(2+) channels that are sensitive to agents that inhibit capacitative Ca(2+) entry and are abolished in the absence of extracellular Ca(2+). Okadaic acid, an inhibitor of PP1 and PP2A phosphatases, blocked the increase in [Ca(2+)](i) produced by the inhibitors of protein kinase A and C. The combined results indicate that the capacitative Ca(2+) entry channels in prothoracic gland cells of B. mori are probably modulated by protein kinases A and C.     

Immunocytochemical evidence for translocation of protein kinase C in human megakaryoblastic leukemic cells: synergistic effects of Ca2+ and activators of protein kinase C on the plasma membrane association

Immunological analysis using monoclonal antibodies against subspecies of protein kinase C revealed the predominant expression of the isozyme, type II, in human megakaryoblastic leukemic cells. We investigated the effects of phorbol diester 12-O-tetradecanoyl phorbol-13-acetate (TPA), the Ca2+ ionophore ionomycin and synthetic diacylglycerol 1-oleoyl-2-acetylglycerol (OAG) on the immunocytochemical localization of protein kinase C in these cells. Indirect immunofluorescence techniques revealed the enzyme to be located in a diffuse cytosolic pattern, in the intact cells. When the cells were exposed to 100 nM TPA, the immunofluorescent staining was translocated from the cytoplasm to the plasma membrane. The translocation was protracted and staining on the membrane decreased in parallel with the Ca2+, phospholipid-dependent protein kinase activity. Treatment of the cells with 500 nM ionomycin caused an apparent translocation comparable with that seen with TPA, however, this translocation was transient and most of the cytosolic staining was within 60 min. We also found that 30 micrograms/ml OAG did not have significant effects on distribution of the staining, but rather acted synergistically on the translocation with the suboptimal concentration of 100 nM ionomycin. A similar synergism was also observed with 10 nM TPA and 100 nM ionomycin. These results obtained in situ provide evidence that intracellular Ca2+ and diacylglycerol regulate membrane binding of the enzyme in vivo.     

Parathyroid hormone activation of map kinase in rat duodenal cells is mediated by 3',5'-cyclic AMP and Ca(2+)

In a previous study, we demonstrated that parathyroid hormone (PTH) stimulates in rat duodenal cells (enterocytes) the phosphorylation and activity of extracellular signal-regulated mitogen-activated protein kinase (MAPK) isoforms ERK1 and ERK2. As PTH activates adenylyl cyclase (AC) and phospholipase C and increases intracellular Ca(2+) in these cells, in the present study we evaluated the involvement of cAMP, Ca(2+) and protein kinase C (PKC) on PTH-induced MAPK activation. We found that MAPK phosphorylation by the hormone did not depend on PKC activation. PTH response could, however, be mimicked by addition of forskolin (5-15 microM), an AC activator, or Sp-cAMP (50-100 microM), a cAMP agonist, and suppressed to a great extent by the AC inhibitor, compound Sq-22536 (0.2-0.4 mM) and the cAMP antagonist Rp-cAMP (0.2 mM). Removal of external Ca(2+) (EGTA 0.5 mM), chelation of intracellular Ca(2+) with BAPTA (5 microM), or blockade of L-type Ca(2+)-channels with verapamil (10 microM) significantly decreased PTH-activation of MAPK. Furthermore, a similar degree of phosphorylation of MAPK was elicited by the Ca(2+) mobilizing agent thapsigargin, the Ca(2+) ionophore A23187, ionomycin and membrane depolarization with high K(+). Inclusion of the calmodulin inhibitor fluphenazine (50 microM) did not prevent hormone effects on MAPK. Taken together, these results indicate that cAMP and Ca(2+) play a role upstream in the signaling mechanism leading to MAPK activation by PTH in rat enterocytes. As Ca(2+) and cAMP antagonists did not block totally PTH-induced MAPK phosphorylation, it is possible that linking of the hormone signal to the MAPK pathway may additionally involve Src, which has been previously shown to be rapidly activated by PTH. Of physiological significance, in agreement with the mitogenic role of the MAPK cascade, PTH increased enterocyte DNA synthesis, and this effect was blocked by the specific inhibitor of MAPK kinase (MEK) PD098059, indicating that hormone modulation of MAPK through these messenger systems stimulates duodenal cell proliferation.     

Role of protein kinase C in catecholamine secretion from digitonin-permeabilized bovine adrenal medullary cells

The effects of staurosporine and K-252a, potent inhibitors of protein kinases, and 12-O-tetradecanoylphorbol-13-acetate (TPA) on catecholamine secretion and protein phosphorylation in digitonin-permeabilized bovine adrenal medullary cells were investigated. Staurosporine and K-252a (0.01-10 microM) did not cause large changes in catecholamine secretion evoked by Ca2+ in digitonin-permeabilized cells whereas these compounds strongly prevented TPA-induced enhancement of catecholamine secretion in a concentration-dependent manner. Incubation of digitonin-permeabilized cells with [gamma-32P]ATP resulted in 32Pi incorporation into a large number of proteins, detected as several major bands and darkened background in autoradiograms. Ca2+ and TPA increased phosphorylation of these proteins. Staurosporine and K-252a markedly inhibited Ca(2+)-induced and TPA-induced increases in protein phosphorylation as well as basal (0 Ca2+) protein phosphorylation in digitonin-permeabilized cells. Long term treatment (24 h) of adrenal medullary cells with 1 microM TPA markedly decreased total cellular protein kinase C activity to about 5.3% of control. Pretreatment of the cells with 1 microM TPA strongly inhibited the TPA-induced enhancement of catecholamine secretion whereas it did not cause large changes in total cellular catecholamine amounts, Ca(2+)-induced catecholamine secretion, and cAMP-induced enhancement of catecholamine secretion from digitonin-permeabilized cells. From these results we conclude that protein kinase C plays a modulatory role in catecholamine secretion rather than being essential for initiating catecholamine secretion.     

An oxidized metabolite of linoleic acid increases intracellular calcium in rat adrenal glomerulosa cells

EKODE, an epoxy-keto derivative of linoleic acid, was previously shown to stimulate aldosterone secretion in rat adrenal glomerulosa cells. In the present study, we investigated the effect of exogenous EKODE on cytosolic [Ca(2+)] increase and aimed to elucidate the mechanism involved in this process. Through the use of the fluorescent Ca(2+)-sensitive dye Fluo-4, EKODE was shown to rapidly increase intracellular [Ca(2+)] ([Ca(2+)](i)) along a bell-shaped dose-response relationship with a maximum peak at 5 microM. Experiments performed in the presence or absence of Ca(2+) revealed that this increase in [Ca(2+)](i) originated exclusively from intracellular pools. EKODE-induced [Ca(2+)](i) increase was blunted by prior application of angiotensin II, Xestospongin C, and cyclopiazonic acid, indicating that inositol trisphosphate (InsP(3))-sensitive Ca(2+) stores can be mobilized by EKODE despite the absence of InsP(3) production. Accordingly, EKODE response was not sensitive to the phospholipase C inhibitor U-73122. EKODE mobilized a Ca(2+) store included in the thapsigargin (TG)-sensitive stores, although the interaction between EKODE and TG appears complex, since EKODE added at the plateau response of TG induced a rapid drop in [Ca(2+)](i). 9-oxo-octadecadienoic acid, another oxidized derivative of linoleic acid, also increases [Ca(2+)](i), with a dose-response curve similar to EKODE. However, arachidonic and linoleic acids at 10 microM failed to increase [Ca(2+)](i) but did reduce the amplitude of the response to EKODE. It is concluded that EKODE mobilizes Ca(2+) from an InsP(3)-sensitive store and that this [Ca(2+)](i) increase is responsible for aldosterone secretion by glomerulosa cells. Similar bell-shaped dose-response curves for aldosterone and [Ca(2+)](i) increases reinforce this hypothesis.     

Protein kinase C evokes quantal catecholamine release from PC12 cells via activation of L-type Ca2+ channels

Application of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to PC12 cells under resting conditions evoked quantal catecholamine secretion, as detected amperometrically. This effect was not mimicked by 4alpha-phorbol-12,13-didecanoate, another phorbol ester, which is inactive with respect to protein kinase C activation, and was prevented by the protein kinase C inhibitor bisindolylmaleimide. TPA also caused a rise of [Ca(2+)](i) in Fura-2-loaded PC12 cells, and again this was not mimicked by 4alpha-phorbol-12,13-didecanoate and could be blocked by bisindolylmaleimide. TPA-evoked secretion was entirely dependent on extracellular Ca(2+) and was fully abolished by nifedipine, as were TPA-induced rises of [Ca(2+)](i). Resting membrane potential, monitored using perforated patch recordings, was unaffected by TPA. However, a small (6-8 mV) hyperpolarizing shift in the voltage dependence of Ca(2+) channel currents (determined using whole-cell patch clamp recordings) was induced by TPA, and this could be fully prevented by nifedipine. In contrast to results with depolarizing stimuli, which evoke exocytosis because of Ca(2+) influx through N-type channels in these cells, the present results indicate that protein kinase C activation leads directly to quantal catecholamine secretion in the absence of depolarizing stimuli via a selective shift in the activation of L-type Ca(2+) channels.     

Staurosporine,a non-specific PKC inhibitor,induces keratinocyte differentiation and raises intracellular calcium,but Ro31–8220, a specific inhibitor,does not

The responsiveness of normal human keratinocytes to different modulators of protein kinase C (PKC) was investigated. The PKC agonist TPA, staurosporine (a non-specific inhibitor), and Ro31–8220 (a specific inhibitor) were studied for effect on cell morphology, growth rate, involucrin expression, and intracellular calcium levels. Surprisingly the response to nanomolar concentrations of staurosporine was similar to TPA and induced a fusiform morphology, inhibited growth, increased involucrin levels, and raised intracellular calcium. Staurosporine also increased the number of cornified envelopes, and its action therefore appeared identical to TPA. In contrast, Ro31–8220 had little effect on morphology or growth and blocked both the TPA-induced growth inhibition and calcium rise. Ro31–8220 had no effect on staurosporine-induced growth inhibition but partially reduced its associated calcium rise. These results suggest PKC activation is required for keratinocyte differentiation and that staurosporine acts like a PKC agonist to give a similar effect as TPA. Specific inhibition of PKC by Ro31–8220 inhibits TPA-induced differentiation. © 1994 wiley-Liss, Inc.     

Protein kinase C stimulates adenylate cyclase activity in prolactin-secreting rat adenoma (GH4C1) pituicytes by inactivating the inhibitory GTP-binding protein Gi

The phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA) and thyroliberin exerted additive stimulatory effects on prolactin release and synthesis in rat adenoma GH4C1 pituicytes in culture. Both TPA and thyroliberin activated the adenylate cyclase in broken cell membranes. When combined, the secretagogues displayed additive effects. TPA did not alter the time course (time lag) of adenylate cyclase activation by hormones, guanosine 5'-[beta,gamma-imino]triphosphate or forskolin, nor did it affect the enzyme's apparent affinity (basal, 7.2 mM; thyroliberin-enhanced, 2.2 mM) for free Mg2+. The TPA-mediated adenylate cyclase activation was entirely dependent on exogenously added guanosine triphosphate. ED50 (dose yielding half-maximal activation) was 60 microM. Access to free Ca2+ was necessary to express TPA activation of the enzyme, however, the presence of calmodulin was not mandatory. TPA-stimulated adenylate cyclase activity was abolished by the biologically inactive phorbol ester, 4 alpha-phorbol didecanoate, by the protein kinase C inhibitor polymyxin B and by pertussis toxin, while thyroliberin-sensitive adenylate cyclase remained unaffected. Experimental conditions known to translocate protein kinase C to the plasma membrane and without inducing adenylate cyclase desensitization, increased both basal and thyroliberin-stimulated enzyme activities, while absolute TPA-enhanced adenylate cyclase was maintained. Association of extracted GTP-binding inhibitory protein, Gi, from S49 cyc- murine lymphoma cells with GH4C1 cell membranes yielded a reduction of basal and hormone-stimulated adenylate cyclase activities, while net inhibition of the cyclase of somatostatin was dramatically enhanced. However, TPA restored completely basal and hormone-elicited adenylate cyclase activities in the Gi-enriched membranes. Finally, TPA completely abolished the somatostatin-induced inhibition of adenylate cyclase in both hybrid and non-hybrid membranes. These data suggest that, in GH4C1 cells, protein kinase C stimulation by phorbol esters completely inactivates the n alpha i subunit of the inhibitory GTP-binding protein, leaving the n beta subunit functionally intact. It can also be inferred that thyroliberin conveys its main effect on the adenylate cyclase through activation of the stimulatory GTP-binding protein, Gs.     

Immunogold electron microscopic demonstration of distinct submembranous localization of the activated gammaPKC depending on the stimulation.

We examined the precise intracellular translocation of gamma subtype of protein kinase C (gammaPKC) after various extracellular stimuli using confocal laser-scanning fluorescent microscopy (CLSM) and immunogold electron microscopy. By CLSM, treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in a slow and irreversible accumulation of green fluorescent protein (GFP)-tagged gammaPKC (gammaPKC-GFP) on the plasma membrane. In contrast, treatment with Ca(2+) ionophore and activation of purinergic or NMDA receptors induced a rapid and transient membrane translocation of gammaPKC-GFP. Although each stimulus resulted in PKC localization at the plasma membrane, electron microscopy revealed that gammaPKC showed a subtle but significantly different localization depending on stimulation. Whereas TPA and UTP induced a sustained localization of gammaPKC-GFP on the plasma membrane, Ca(2+) ionophore and NMDA rapidly translocated gammaPKC-GFP to the plasma membrane and then restricted gammaPKC-GFP in submembranous area (<500 nm from the plasma membrane). These results suggest that Ca(2+) influx alone induced the association of gammaPKC with the plasma membrane for only a moment and then located this enzyme at a proper distance in a touch-and-go manner, whereas diacylglycerol or TPA tightly anchored this enzyme on the plasma membrane. The distinct subcellular targeting of gammaPKC in response to various stimuli suggests a novel mechanism for PKC activation.     

Recent breakthroughs in the molecular mechanism of capacitative calcium entry (with thoughts on how we got here)

Activation of phospholipase C by G-protein-coupled receptors results in release of intracellular Ca(2+) and activation of Ca(2+) channels in the plasma membrane. The intracellular release of Ca(2+) is signaled by the second messenger, inositol 1,4,5-trisphosphate. Ca(2+) entry involves signaling from depleted intracellular stores to plasma membrane Ca(2+) channels, a process referred to as capacitative calcium entry or store-operated calcium entry. The electrophysiological current associated with capacitative calcium entry is the calcium-release-activated calcium current, or I(crac). In the 20 years since the inception of the concept of capacitative calcium entry, a variety of activation mechanisms have been proposed, and there has been considerable interest in the possibility of transient receptor potential channels functioning as store-operated channels. However, in the past 2 years, two major players in both the signaling and permeation mechanisms for store-operated channels have been discovered: Stim1 (and possibly Stim2) and the Orai proteins. Activation of store-operated channels involves an endoplasmic reticulum Ca(2+) sensor called Stim1. Stim1 acts by redistributing within a small component of the endoplasmic reticulum, approaching the plasma membrane, but does not appear to translocate into the plasma membrane. Stim1, either directly or indirectly, signals to plasma membrane Orai proteins which constitute pore-forming subunits of store-operated channels.     

Cholecystokinin-evoked Ca(2+) waves in isolated mouse pancreatic acinar cells are modulated by activation of cytosolic phospholipase A(2), phospholipase D, and protein kinase C.

We employed confocal laser-scanning microscopy to monitor cholecystokinin (CCK)-evoked Ca(2+) signals in fluo-3-loaded mouse pancreatic acinar cells. CCK-8-induced Ca(2+) signals start at the luminal cell pole and subsequently spread toward the basolateral membrane. Ca(2+) waves elicited by stimulation of high-affinity CCK receptors (h.a.CCK-R) with 20 pM CCK-8 spread with a slower rate than those induced by activation of low-affinity CCK receptors (l.a. CCK-R) with 10 nM CCK-8. However, the magnitude of the initial Ca(2+) release was the same at both CCK-8 concentrations, suggesting that the secondary Ca(2+) release from intracellular stores is modulated by activation of different intracellular pathways in response to low and high CCK-8 concentrations. Our experiments suggest that the propagation of Ca(2+) waves is modulated by protein kinase C (PKC) and arachidonic acid (AA). The data indicate that h.a. CCK-R are linked to phospholipase C (PLC) and phospholipase A(2) (PLA(2)) cascades, whereas l.a.CCK-R are coupled to PLC and phospholipase D (PLD) cascades. The products of PLA(2) and PLD activation, AA and diacylglycerol (DAG), cause inhibition of Ca(2+) wave propagation by yet unknown mechanisms.