Cell Tissue Res DOI 10.1007/s00441-015-2199-3

REGULAR ARTICLE

Expression and localization of the diacylglycerol kinase family and of phosphoinositide signaling molecules in adrenal gland Yasukazu Hozumi 1 & Ryo Akimoto 1 & Akihito Suzuki 2 & Koichi Otani 2 & Masahiko Watanabe 3 & Kaoru Goto 1

Received: 27 October 2014 / Accepted: 13 April 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Adrenal glands play a central role in the secretion of steroid hormones and catecholamines. Previous studies have revealed that molecules engaged in phosphoinositide (PI) turnover are expressed in the adrenal gland, suggesting the importance of PI signaling in adrenal signal transduction. Diacylglycerol kinase (DGK) catalyzes the phosphorylation of diacylglycerol (DG), a major second messenger in the PI signaling cascade. The DGK family is expressed in distinct patterns in endocrine organs at the mRNA and protein levels. Nevertheless, little is known about the characteristics and morphological aspects of DGKs in the adrenal gland. We have performed immunohistochemical analyses to investigate the expression and localization of DGK isozymes, together with PI signaling molecules, in the adrenal gland at the protein level. Our results show that the DGK family and a set of PI signaling molecules are expressed intensely in zona glomerulosa cells and medullary chromaffin cells in the adrenal gland. In adrenal cells, DGKγ localizes to the Golgi complex, DGKε to the plasma membrane, and DGKζ to the This work was supported by Grants-in-Aid from The Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (Y.H., K.G.). Electronic supplementary material The online version of this article (doi:10.1007/s00441-015-2199-3) contains supplementary material, which is available to authorized users. * Yasukazu Hozumi [email protected] 1

Department of Anatomy and Cell Biology, Yamagata University School of Medicine, Iida-nishi 2-2-2, Yamagata 990-9585, Japan

2

Department of Psychiatry, Yamagata University School of Medicine, Yamagata 990-9585, Japan

3

Department of Anatomy, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan

nucleus. These findings show the distinct expression and subcellular localization of DGK isozymes and PI signaling molecules in the adrenal gland, suggesting that each DGK isozyme has a role in signal transduction in adrenal cells, especially in the zona glomerulosa and medulla. Keywords Diacylglycerol kinase . Phosphoinositide turnover . Zona glomerulosa . Medulla . Immunohistochemistry . Rat (Wistar, male)

Introduction In response to external stimuli, Gq protein-coupled receptors trigger activation of phospholipase C (PLC), which yields a pair of second messengers, diacylglycerol (DG) and inositol 1, 4,5-trisphosphate (IP3), during phosphoinositide (PI) turnover (Cockcroft and Thomas 1992; Rhee et al. 1989). In this system, diacylglycerol kinase (DGK) phosphorylates DG to produce phosphatidic acid (PA; Kanoh et al. 1990). Awell-known functional role of DGK is the regulation of protein kinase C (PKC), for which DG acts as an allosteric activator. The activity of PKC plays a central function in various cell types (Martelli et al. 2004; Nishizuka 1992; Ron and Kazanietz 1999). Moreover, recent studies have revealed that PA, the product of DGK, acts as a messenger to regulate other signaling molecules (Merida et al. 2008; Topham 2006; Zhang and Du 2009). Therefore, DGK is thought to mediate signal transduction by modulating the levels of DG and PA, i.e., the attenuation of DG and the production of PA signals. To date, 10 mammalian DGK isozymes have been identified (Goto et al. 2007; Martelli et al. 2011; Sakane et al. 2007; Topham 2006). A salient feature of the DGK family is that most isozymes are expressed abundantly in the brain, suggesting the physiological importance of this enzyme family for proper brain

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function (Goto and Kondo 2004; Goto et al. 2006). Additionally, we have previously shown that the DGK family is expressed in distinct patterns in the pituitary gland (Hozumi et al. 2010). Recent studies have revealed that PI signaling molecules, including the Gq subclass of the G protein, PLCβ subfamily, and conventional PKCs, are expressed at the protein level in adrenal glands (Cote et al. 1997; O'Sullivan and Bunn 2006). Reportedly, analysis by reverse transcription plus the polymerase chain reaction (RT-PCR) has revealed several DGKs in human adrenocortical-cell-derived H295R cells: DGKδ and DGKη abundantly, and DGKι and DGKζ weakly (Li et al. 2007). These results imply that the DGK family participates in PI signaling in the adrenal gland, although little is known about the nature and morphological aspects of DGKs in this endocrine organ. We have raised specific antibodies against DGKs and have examined unique expression patterns of the respective isozymes in the rat central and peripheral nervous system, heart, vascular smooth muscle and endothelial cells, lung, liver, female reproductive organs, pituitary gland, and retina under pathophysiological conditions (Goto et al. 2007, 2014; Hozumi and Goto 2012; Hozumi et al. 2013; Nakano et al. 2009, 2012). In the study described herein, we have investigated the expression and localization of DGK isozymes and PI signaling molecules at the protein level in the adrenal gland by using immunohistochemistry with a battery of wellcharacterized antibodies. Our results show that isozymes of the DGK family display distinct cellular expression and subcellular localization patterns in adrenal cells. These findings suggest a unique role for each isozyme in signal transduction of this organ.

Materials and methods Animals Adult male Wistar rats (Japan SLC) were used in the present study. Rats were housed under freely ranging conditions in our animal facilities under normal lighting conditions (lights on 08:00–20:00) and were allowed ad libitum access to normal rodent chow and water. All animals were treated according to the guidelines for the care and use of laboratory animals of the Yamagata University School of Medicine. Immunoblot analysis Adrenal glands of 9-week-old rats were homogenized with 4 volumes of a buffer containing 10 mM TRIS–HCl (pH 7.4), 20 mM KCl, 0.1 mM EDTA, and 0.25 M sucrose and centrifuged at 1000g for 10 min at 4 °C to remove debris. Protein concentration was determined by using BCA Protein Assay

Reagent (Thermo Scientific, Rockford, Ill., USA). Values were the means of triplicate determinations. The resulting supernatant (30 μg) was boiled for 5 min in sodium dodecylsulfate (SDS) sample buffer (New England Biolabs, Beverly, Mass. USA) and subjected to 10 % SDSpolyacrylamide gel electrophoresis (PAGE). The proteins were then electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, Mass., USA). After the blocking of the non-specific binding sites with 4 % non-fat dry milk (w/v) in phosphate-buffered saline (PBS) containing 0.02 % sodium azide and 0.2 % Tween 20, the membrane was incubated overnight at 4 °C with rabbit anti-DGK antibodies (0.5 μg/ml; Supplemental Table S1) in 2 % non-fat dry milk (w/v) in PBS containing 0.02 % sodium azide and 0.1 % Tween 20. Sites of antigen-antibody reaction were visualized by using the chemiluminescent Immobilon Western blotting detection system (Millipore). We repeated the immunoblot three times and obtained the same results from those experiments. Tissue and section preparation For immunohistochemistry, rats anesthetized with ether were fixed transcardially with 4 % paraformaldehyde in 0.1 M sodium phosphate buffer (PB, pH 7.2; Hozumi et al. 2013). After fixation, adrenal glands were cryoprotected in 30 % sucrose/0.1 M PB and cut into 16-μm-thick sections with a cryostat (CM1900; Leica, Nussloch, Germany). Immunohistochemistry A brief pre-treatment with methanol greatly enhanced immunoreactivities for PI signaling molecules (Hozumi et al. 2008, 2013). Therefore, sections for immunofluorescence were treated, prior to normal serum blocking, by dipping in 100 % methanol for 10 min. All immunohistochemical incubations were performed at room temperature (~18 °C). For double-immunofluorescence, rabbit anti-DGKα antibody (1 μg/ml; Goto et al. 1992), rabbit anti-DGKβ antibody (1 μg/ml; Hozumi et al. 2008), rabbit anti-DGKγ antibody (1 μg/ml; Nakano et al. 2012), rabbit anti-DGKε antibody (1 μg/ml; Nakano et al. 2009), rabbit anti-DGKζ antibody (1 μg/ml; Hozumi et al. 2003), rabbit anti-DGKι antibody (1 μg/ml; Ito et al. 2004), rabbit anti-G protein alpha(q)/alpha11 (anti-Gαq/11; 1 μg/ml; sc-392; Santa Cruz Biotechnology, Santa Cruz, Calif., USA), mouse antiphospholipase Cβ1 (PLCβ1; 1 μg/ml; sc-5291; Santa Cruz Biotechnology), goat anti-phospholipase Cβ2 (PLCβ2; 1 μg/ml; sc-31757; Santa Cruz Biotechnology), rabbit antiphospholipase Cβ3 (PLCβ3; 1 μg/ml; Nomura et al. 2007), rabbit anti-phospholipase Cβ4 (PLCβ4; 1 μg/ml; Nakamura et al. 2004), rabbit anti-protein kinase Cα (PKCα; 1 μg/ml; Frontier Institute, Hokkaido, Japan), rabbit anti-protein kinase

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CβII (PKCβII; 1 μg/ml; Hasegawa et al. 2008), or rabbit antiprotein kinase Cγ (PKCγ; 1 μg/ml; Yoshida et al. 2006) diluted with PBS containing 0.1 % Triton X-100 was incubated overnight in a mixture with goat anti-calregulin (1 μg/ml; sc6468; Santa Cruz Biotechnology), mouse anti-ED2 (1:100; MCA342GA; AbD Serotec, Oxford, UK), mouse anti-Golgi 58 K (1:100; G2404; Sigma, St. Louis, Mo., USA), guinea pig anti-phenylethanolamine-N-methyltransferase (PNMT; 1:200; PS256; Monosan, Uden, Netherlands), or mouse antityrosine hydroxylase (TH; 1:1000; MAB318; Chemicon, Temecula, Calif., USA). After thorough washes in PBS, the antibodies were visualized by a 2-h incubation with speciesspecific secondary antibodies at a dilution of 1:200 (Molecular Probes, Eugene, Ore., USA). The specificities of the antibodies used in the current study are shown in Supplemental Table S1. The nuclei of cells were stained by using 4,6diamidino-2-phenylindole (DAPI; Molecular Probes) and scanned by using a confocal laser scanning microscope (LSM700, Carl Zeiss, Göttingen, Germany) in multitrack mode.

Results Expression of DGK isozymes in adrenal gland We first confirmed that DGK isozymes, including DGKα, DGKγ, DGKε, DGKζ, and DGKι, were expressed at the protein level in the adrenal gland (Fig. 1). We then proceeded to examine the cellular expression and localization of these DGKs and of other PI signaling molecules in the adrenal gland by using immunofluorescence microscopy. We also employed

Fig. 1 Immunoblot analysis of diacylglycerol kinase (DGK) isozymes in adrenal gland. Single bands with the molecular mass of DGKα, DGKγ, DGKε, DGKζ, or DGKι were detected by immunoblot analysis with the antibodies against DGKs in lysates (30 μg) of rat adrenal glands. The position of the standard protein marker is indicated left

DAPI (for the nuclei) and organelle markers to delineate subcellular localizations. Intense DGK immunoreactivities were found in the zona glomerulosa and medulla, although DGKζ and DGKι were principally detected throughout all cortical layers (Fig. 2), and DGKα was diffusely and weakly expressed throughout the cortex and medulla (data not shown). In the following experiments, we specifically examined the zona glomerulosa and medulla by using detailed analyses.

Zona glomerulosa Of the DGK isozymes, immunoreactivities for DGKγ, −ε, −ζ, and –ι were found intensely in the zona glomerulosa (Fig. 2). DGKγ immunoreactivity was localized to the juxtanuclear position as a granular structure in zona glomerulosa cells (Fig. 3a). Because this image closely resembled that of the Golgi complex, we performed double-immunolabeling experiments by using an antibody against Golgi 58 K, a marker for the Golgi complex. DGKγ immunoreactivity was mostly colocalized with the Golgi marker staining (Fig. 3a). Immunohistochemical signals for DGKε were regarded as tiny puncta on or close to the plasma membrane (Fig. 3b). Because our previous study showed that DGKε localized to the endoplasmic reticulum (ER; Matsui et al. 2014), we conducted double-immunolabeling experiments with an antibody against calregulin, a marker for the ER. Puncta labeled for DGKε were only partly colocalized with calregulin staining (Fig. 3b). These puncta appeared to outline the boundary between the cells, suggesting that DGKε was localized to the

Fig. 2 Distribution of DGK isozymes in the adrenal gland. Low magnification images of immunofluorescence for DGK isozymes in the adrenal gland. Intense labeling for DGK isozymes is observed in the zona glomerulosa and medulla (ZG zona glomerulosa, ZF zona fasciculata, ZR zona reticularis, Me medulla). Bars 100 μm

Cell Tissue Res Fig. 3 Expression and localization of DGK isozymes in the zona glomerulosa. Immunofluorescence for characterization of DGKγ (a), DGKε (b), DGKζ (c), and DGKι (d) in the zona glomerulosa. DGKγ immunoreactivity is localized to the juxtanuclear position as a granular structure (a). Note that the structure labeled for DGKγ overlaps with immunostaining for Golgi 58 K (Golgi marker). Immunohistochemical signals for DGKε are detectable as tiny puncta on or close to the plasma membrane and are only partly colocalized with calregulin staining (endoplasmic reticulum marker; b). DGKζ immunoreactivity is observed in the nucleus of zona glomerulosa cells (c). Immunolabeling for DGKι is distributed throughout the cytoplasm in zona glomerulosa cells (d). Blue fluorescence represents nuclear staining with 4,6-diamidino-2-phenylindole (DAPI). Bars 10 μm

plasma membrane. DGKζ immunoreactivity was observed in the nucleus of zona glomerulosa cells (Fig. 3c). Immunolabeling for DGKι was distributed throughout the cytoplasm in zona glomerulosa cells (Fig. 3d). Of the PI signaling molecules, intense signals for Gαq/11 (Fig. 4a) and PLCβ1 (Fig. 4b) were visible in the plasma membrane of zona glomerulosa cells, but those for PLCβ3 (Fig. 4c), PKCα (Fig. 4d), and PKCβII (Fig. 4e) could only be weakly recognized in the cytoplasm of some, but not all, glomerulosa cells. Medulla Immunoreactivities for DGKα, −γ, −ε, −ζ, and -ι were found in the medulla (Figs. 2, 5). Immunohistochemical signals for DGKα were visible weakly in the cytoplasm of medullary chromaffin cells labeled with an antibody to tyrosine hydroxylase (TH; O'Sullivan and Bunn 2006; Fig. 5a). In detail, DGKγ immunoreactivity was localized intensely to the juxtanuclear position as a granular structure in chromaffin cells (Fig. 5b). DGKγ immunoreactivity was

mostly colocalized with Golgi marker staining (Fig. 5c), similar to the results in the zona glomerulosa cells. DGKζ immunoreactivity was detected in the nucleus of TH-positive chromaffin cells (Fig. 5d). DGKι immunoreactivity was evident in the cytoplasm of TH-positive chromaffin cells (Fig. 5e). In addition, immunolabeling for DGKι was shown to be associated with fine nerve fibers running within the medulla (arrowheads in Fig. 5e). In contrast, immunohistochemical signals for DGKε were observed strongly in elongated THnegative non-chromaffin cells (arrows in Fig. 5f), although no significant DGKε immunolabeling was detected in TH-positive chromaffin cells. On the other hand, double-immunolabeling with an antibody against ED2 (a marker for medullary macrophages; Schober et al. 1998) revealed that DGKε immunoreactivity occurred in ED2positive cells (Fig. 5g), suggesting that DGKε was expressed in medullary macrophages. Next, in order to determine the presence of DGK in chromaffin cells containing adrenaline or noradrenaline, we performed tripleimmunostaining with antibodies against TH and

Cell Tissue Res Fig. 4 Expression and localization of phosphoinositide (PI) signaling molecules in the zona glomerulosa (Gαq/11 G protein alpha(q)/alpha11, PLC phospholipase C, PKC protein kinase C). Immunofluorescence for characterization of Gαq/11 (a), PLCβ1 (b), PLCβ3 (c), PKCα (d), and PKCβII (e) in the zona glomerulosa. Intense signals for Gαq/11 (a) and PLCβ1 (b) are visible in the plasma membrane of zona glomerulosa cells, but those for PLCβ3 (c), PKCα (d), and PKCβII (e) are only weakly detected in the cytoplasm. Blue fluorescence represents nuclear staining with DAPI. Bars 10 μm

phenylethanolamine-N-methyltransferase (PNMT), together with DGKγ, DGKζ, or DGKι, which were expressed intensely in chromaffin cells. TH-positive chromaffin cells are divisible into two groups: noradrenaline cells immunoreactive to dopamine β-hydroxylase (DBH) (TH in this study) alone and adrenaline cells immunoreactive to both DBH (or TH) and PNMT (Oomori et al. 1998). Interestingly, immunoreactivities for DGKγ (Fig. 5h) and DGKζ (Fig. 5i) were detected in TH-positive/PNMT-positive adrenaline cells, but not in TH-positive/PNMT-negative noradrenaline cells. On the other hand, DGKι immunoreactivity was observed in both TH-positive/PNMT-positive adrenaline cells and TH-positive/PNMT-negative noradrenaline cells (Fig. 5j). Of the PI signaling molecules, immunoreactivities for Gαq/11, PLCβ3, PLCβ4, PKCα, and PKCβII were detected moderately to intensely in the medulla (Fig. 6). Immunohistochemical signals for Gαq/11 (Fig. 6a), PLCβ4 (Fig. 6b), and PKCα (Fig. 6c) were detected in the cytoplasm of TH-positive chromaffin cells, but those for PLCβ3 (Fig. 6d) and PKCβII (Fig. 6e) were below the detection level. However, abundant expression of PLCβ3 (Fig. 6d) and PKCβII (Fig. 6e) was observed in TH-negative cell cytoplasm. PLCβ3 was detected in large ovoid cells (arrow in Fig. 6d) and fine nerve fibers running within the medulla (arrowheads in Fig. 6d), whereas PKCβII was found in small cells with irregularly shaped morphologies (arrows in Fig. 6e), as in DGKε-positive cells. Double-immunolabeling with an antibody against ED2 revealed that PKCβII immunoreactivity occurred in ED2-positive cells (Fig. 6f),

suggesting that PKCβII was expressed in medullary macrophages.

Discussion This study has revealed, for the first time, the expression and localization of DGKs in the adrenal gland at the protein level by using specific antibodies. We show clearly that DGK isozymes exhibit distinct localization patterns in rat adrenal gland. These findings suggest that DGKs are involved in adrenal signal transduction, and that they play different roles in the distinct adrenal cells. Tables 1 and 2 present the distribution of DGKs and PI signaling molecules in rat adrenal glands. This study has demonstrated that DGKs and a set of PI signaling molecules are expressed intensely in zona glomerulosa and medullary chromaffin cells, although expression in the zona fasciculata and reticularis is weak. Our findings suggest that the PI signaling cascade is actively operating in these regions of the adrenal gland. What is the functional involvement of PI signaling cascade in zona glomerulosa and medullary chromaffin cells? Of the hormones acting on the adrenal gland, Angiotensin II (Ang II), the central product of the renin-angiotensin system, is intimately involved in the regulatory mechanism of systemic and local blood pressure via its vasoconstrictive effect, thereby influencing renal tubules to retain sodium and water and stimulating aldosterone release from the adrenal gland (Timmermans et al. 1993). This octapeptide hormone Ang II acts on

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peripheral target organs and tissues through its receptors (Mendelsohn et al. 1990). The Ang II receptor has been classified into two main subtypes, designated as AT1 and AT 2 (Murphy et al. 1991; Timmermans et al. 1992; Tsuzuki et al. 1994). AT1 receptor is distributed ubiquitously and abundantly in adult tissues, including blood vessels, heart, kidney, adrenal gland, liver, brain, and lung (Timmermans et al. 1993). On the other hand, AT2 receptor is expressed in small-/medium-sized cultured neurons of human and rat dorsal root ganglion (Anand et al. 2013). Recently, the involvement of AT2 receptor in pain signaling has been revealed, and EMA401, a highly selective AT2 receptor antagonist, is under development as a novel neuropathic pain therapeutic agent (Danser and Anand 2014; Rice et al. 2014). These data suggest that both AT1 and AT2 receptors mediate many physiological actions in mammalian organs. In a signaling cascade, the AT1 receptor is a member of the seven transmembrane-spanning, G proteincoupled receptor family. It binds to heterotrimeric G proteins and lacks intrinsic tyrosine kinase activity (Kim and Iwao 2000). Of particular interest is that, reportedly, AT1 receptors of cardiac myocytes and vascular smooth muscle cells couple to a heterotrimeric G protein, Gq (Berk and Corson 1997; Bernstein et al. 1998; Griendling et al. 1997; Kim and Iwao 2000; Schieffer et al. 1996). In addition, DG-sensitive enzymes, such as PKC and serine/threonine kinase protein kinase D (PKD), are thought to be important in aldosterone secretion (Shapiro et al. 2010; Wang 2006), suggesting that DGK is involved in the Ang II signaling cascade by modulating the level of DG. Morphological studies have revealed moderate levels of AT1 receptor mRNA expression in both the zona glomerulosa and medulla of the adrenal gland (Gasc et al. 1994; Johren et al. 1995). Subsequent studies have confirmed that the AT1 receptors are detectable in these regions at the protein level (Frei et al. 2001; Giles et al. 1999). When examined at the ultrastructural level, immunoreaction products against AT1 receptors are restricted to the plasma membranes of cells in the zona glomerulosa (Giles et al. 1999). Collectively, these reports demonstrate that zona glomerulosa cells and chromaffin cells are the primary sites of action of Ang II through AT1 receptors. In this regard, our results show that DGKγ, DGKε, DGKζ, and DGKι are expressed in zona glomerulosa cells, but that they localize to distinct subcellular sites. These findings suggest that at least four DGKs are engaged in functions of zona glomerulosa cells at distinct subcellular sites that operate the AT1 receptor-mediated Ang II signaling cascade and subsequent aldosterone secretion. In a series involving this signal transduction, Gαq/11 and PLCβ1, which localize to the plasma

Fig. 5 Expression and localization of DGK isozymes in the medulla.„ Immunofluorescence for characterization of DGKα (a), DGKγ (b, c, h), DGKζ (d, i), DGKι (e, j), and DGKε (f, g) in the medulla. Immunostaining for tyrosine hydroxylase (TH; chromaffin cell marker), Golgi 58 K, ED2 (medullary macrophage marker), and phenylethanolamineN-methyltransferase (PNMT) is indicated. Immunohistochemical signals for DGKα are detected weakly in the cytoplasm of medullary chromaffin cells (a). DGKγ immunoreactivity is localized intensely to the juxtanuclear position as a granular structure in chromaffin cells (b). DGKγ immunoreactivity is mostly colocalized with Golgi marker staining (c). DGKζ immunoreactivity is localized to the nucleus of TH-positive chromaffin cells (d). DGKι immunoreactivity is detected in the cytoplasm of TH-positive chromaffin cells (e). Immunolabeling for DGKι is also associated with fine nerve fibers running within the medulla (arrowheads in e). Immunohistochemical signals for DGKε are observed intensely in TH-negative non-chromaffin cells (arrows in f). DGKε immunoreactivity is detected in ED2-positive cells (g). Immunoreactivities for DGKγ (h) and DGKζ (i) are solely found in TH-positive/PNMTpositive adrenaline cells, but not in TH-positive/PNMT-negative noradrenaline cells. DGKι immunoreactivity is observed in both TH-positive/PNMT-positive adrenaline cells and TH-positive/PNMT-negative noradrenaline cells (j). Blue fluorescence represents nuclear staining with DAPI (A adrenaline cells, NA noradrenaline cells). Bars 10 μm (a, b, g, h, i, j), 5 μm (c, d), 20 μm (e, f)

membrane, might be involved in the G protein-coupled AT1 receptor, leading to the triggering of the PI signal. However, PLCβ3, which resides in the cytoplasm, might participate downstream of this cascade, presumably leading to aldosterone secretion. Each DGK in the zona glomerulosa cells might regulate the metabolism of DG at distinct sites. Medullary chromaffin cells are a primary output of the sympathetic nervous system, releasing catecholamines into the circulation (Aunis 1998). We have also found immunolabeling of DGKα, DGKγ, DGKζ, and DGKι in medullary chromaffin cells at similar subcellular sites to those in zona glomerulosa cells. In chromaffin cells, sympathetic activity elicits catecholamine release through cholinergic synaptic input from the innervating splanchnic nerve. In this case, acetylcholine (Ach) binds to ionotropic receptors, but not to G protein-coupled muscarinic receptors, on chromaffin cells, thereby triggering a sodium-based action potential that activates highvoltage-activated calcium channels. These reports make it less likely that Gαq-coupled PI signaling is actively engaged in Ach receptors. Therefore, PI signaling is probably involved in the AT1 receptor cascade rather than in Ach receptor signaling in chromaffin cells, as discussed previously. Interestingly, the expression of DGKγ and DGKζ is restricted to adrenaline cells, whereas that of DGKι is observed in both adrenaline and noradrenaline cells. These distinct cellular expression patterns of DGK isozymes in chromaffin cells suggest that a unique role is assigned to each isozyme in catecholamine production.

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Fig. 6 Expression and localization of PI signaling molecules in the medulla. Immunofluorescence for characterization of Gαq/11 (a), PLCβ4 (b), PKCα (c), PLCβ3 (d), and PKCβII (e, f) in the medulla. Green fluorescence represents the chromaffin cell marker tyrosine hydroxylase (TH) and medullary macrophage marker ED2 as indicated. Immunohistochemical signals for Gαq/11 (a), PLCβ4 (b), and PKCα (c) are detected in the cytoplasm of TH-positive chromaffin cells. PLCβ4 immunoreactivity is distributed throughout the medulla (b), but the

intensity is variable among cell clusters, as reported previously (O'Sullivan and Bunn 2006). PLCβ3 is detected in large ovoid cells (arrow in d) and fine nerve fibers running within the medulla (arrowheads in d). PKCβII is found in TH-negative small cells with i r r e g u l a r l y s h a p e d m o r p h o l o g i e s ( a rro ws in e) . PK C β I I immunoreactivity is colocalized with ED2 staining (f). Blue fluorescence represents nuclear staining with DAPI. Bars 20 μm

Reportedly, PLCβ and PKC isozymes are expressed at moderate levels in chromaffin cells (O'Sullivan and Bunn 2006). An early investigation into PLC activity was conducted on tissue from the adrenal medulla (Hokin et al. 1958). Subsequent studies of adrenal medullary slices and isolated adrenal chromaffin cells showed that PLC has an important role in the physiological regulation of this tissue (Bittner and Holz 2005; Bunn and Dunkley 1997; Marley 2003; RobertsThomson et al. 2004). These reports support the idea that the DG-mediated PKC cascade in PI signaling also operates in adrenal chromaffin cells in which the Gαq/11-PLCβ4DGKα and DGKι cascade is detected in the cytoplasm. Notably, DGKε and PKCβII are detected in TH-negative cells, but not in TH-positive chromaffin cells. Non-chromaffin cells in the medulla reportedly represent ganglion cells and

macrophages (Sarria et al. 2006; Schober et al. 1998). In this regard, we have revealed that DGKε and PKCβII are detectable in ED2-positive cells, suggesting that these molecules are expressed in medullary macrophages. Several unanswered questions remain. We suggest that signaling for AT1 receptor is operated via a series of PI-related signaling cascades, including Gαq/11, PLCβ, and DGKs. However, direct evidence for this putative signaling cascade remains insufficient. Further examination must be conducted to establish the cascades by using specific antibodies against AT1 receptors and to explain why more than one DGK isozyme exists in zona glomerulosa cells and chromaffin cells. This question is extremely important not only for adrenal cells, but also for other types of cells, such as neurons (Hozumi et al. 2003, 2009). A key to resolving this question

Cell Tissue Res Table 1 Expression and localization of diacylglycerol kinases (DGK) and of phosphoinositide (PI) signaling molecules (Gαq/11 G protein alpha(q)/ alpha11, PLC phospholipase C, PKC protein kinase C)) in the cortex of rat adrenal gland (− absent, ± weakly present, + present, ++ intense) Molecule Zona Zona Zona Immunohistochemical glomerulosa fasciculata reticularis localization cells cells cells DGKα DGKβ DGKγ DGKε

− − ++ ++

− − ± −

− − + −

DGKζ DGKι Gαq/11 PLCβ1 PLCβ2 PLCβ3 PLCβ4 PKCα PKCβII PKCγ

++ ++ ++ ++ − + − ± ± −

± + − − − − − − − −

+ + ± − − − − − − −

− − Golgi complex Plasma membrane and endoplasmic reticulum Nucleus Cytoplasm Plasma membrane Plasma membrane − Cytoplasm − Cytoplasm Cytoplasm −

is the subcellular localization of these isozymes. Results from the present study have revealed that DGKs localize to distinct

subcellular sites, i.e., DGKα and DGKι to the cytoplasm, DGKγ to the Golgi complex, DGKε to the plasma membrane/ER, and DGKζ to the nucleus. This pattern of DGK subcellular localization is consistent with that found in our previous study (Kobayashi et al. 2007). We infer that each DGK is engaged in DG metabolism at its own subcellular site in order to modulate the cellular activity of distinct cells. Some DGK might be involved in the receptor-stimulated DG signal in the plasma membrane, whereas others might be engaged in the activities of organelles or in DG-involved basal lipid metabolism. Indeed, DGK-knockout mice might provide us with clues to address the significance of these fundamental questions of molecular diversity. Furthermore, several G proteincoupled receptors have been demonstrated to be altered by loss or gain of function mutations, leading respectively to the clinical phenotype of hormone defect or excess (Lania et al. 2001). In particular, mutations of Gαq cDNA occur infrequently, if at all, in human pituitary adenomas (Oyesiku et al. 1997), suggesting the importance of the DG-dependent signaling cascade in endocrine organs. In conclusion, DGK isozymes, together with a set of PI signaling molecules, are expressed broadly in the adrenal gland. Their distinct cellular expression and subcellular localization patterns suggest that each isozyme plays a unique role in adrenal signal transduction. The findings presented herein provide clues for elucidating functional aspects of angiotensin

Table 2 Expression and localization of DGKs and PI signaling molecules in the medulla of rat adrenal gland (− absent, + present, ++ intense) Molecule

Chromaffin cells

Macrophages

Ganglion cells

Nerve fibers

Immunohistochemical localization

Adrenaline cells

Noradrenaline cells

DGKα

Not examined

−

−

−

−

DGKβ DGKγ DGKε DGKζ DGKι

− ++ − ++ ++

− − − − ++

− − ++ − −

− − − − −

− − − − ++

Cytoplasm − Golgi complex Cytoplasm Nucleus Cytoplasm

Molecule

Chromaffin cells

Macrophages

Ganglion cells

Nerve fibers

Immunohistochemical localization

Gαq/11 PLCβ1 PLCβ2 PLCβ3 PLCβ4 PKCα PKCβII PKCγ

+ − − − + to ++ ++ − −

− − − − − − ++ −

− − − ++ − − − −

− − − ++ − − − −

Plasma membrane − − Cytoplasm Cytoplasm Cytoplasm Cytoplasm −

Cell Tissue Res

receptor signaling involved with DGKs in the pathophysiology of the adrenal gland.

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