The cortactin-binding postsynaptic density protein proSAP1 in non-neuronal cells.

Proline-rich synapse-associated protein-1 (ProSAP1) is a neuronal PDZ domain-containing protein that has recently been identified as an essential element of the postsynaptic density. Via its interaction with the actin-binding protein cortactin and its integrative function in the organization of neurotransmitter receptors, ProSAP1 is believed to be involved in the linkage of the postsynaptic signaling machinery to the actin-based cytoskeleton, and may play a role in the cytoskeletal rearrangements that underlie synaptic plasticity. As a result of our ongoing studies on the distribution and function of this novel PDZ domain protein, we now report that the expression of ProSAP1 is restricted neither to neurons and interneuronal junctions nor to the nervous system. Using immunohistochemical techniques in conjunction with specific antibodies, we found that, in the CNS, ProSAP1 can be detected in certain glial cells, such as ependymal cells, tanycytes, subpial/radial astrocytes, and in the choroid plexus epithelium. Moreover, our immunohistochemical analyses revealed the presence of ProSAP1 in endocrine cells of the adenohypophysis and of the pancreas, as well as in non-neuronal cell types of other organs. In the pancreas, ProSAP1 immunoreactivity was also localized in the duct system of the exocrine parenchyma. Our findings demonstrate that, in addition to neurons, ProSAP1 is present in various non-neuronal cells, in which it may play a crucial role in the dynamics of the actin-based cytoskeleton. (J Histochem Cytochem 49:639-648, 2001)

Evidence for microvesicular storage and release of glycine in rodent pinealocytes.

Prompted by previous studies suggesting a regulatory role for the inhibitory amino acid gamma-aminobutyric acid (GABA) within the mammalian pineal gland, we carried out a study of rat and gerbil pineal organs to elucidate whether there is evidence for a vesicular storage and release of GABA and/or glycine. Immunohistochemistry revealed the presence of the vesicular inhibitory amino acid transporter in pinealocytes. Moreover, we found that, in addition to glutamate and aspartate, cultured pinealocytes also released glycine upon stimulation by depolarizing concentrations of KCl, whereas the content of GABA in the culture medium did not exceed the detection limit either under control conditions or following KCl application. Therefore, we propose that glycine is a further component of the paracrine signaling system within the pineal organ which is based on the compartment of synaptic-like microvesicles (SLMVs) inside pinealocytes.

Uteroglobin promoter-targeted c-MYC expression in transgenic mice cause hyperplasia of Clara cells and malignant transformation of T-lymphoblasts and tubular epithelial cells.

To investigate the influence of the proto-oncogene c-MYC on tumor development in different epithelial tissues which secrete Clara Cell Secretory Protein (uteroglobin, UG), transgenic mouse lines were established expressing the human c-MYC proto-oncogene under the control of the rabbit UG-promoter. These mice expressed the c-MYC transgene in Clara cells and other UG expressing tissues like uterus and prostate. In the bronchioalveolar epithelium of the lung hyperplasias developed originating from Clara cells. Surprisingly, transgenics most frequently developed T-lymphoblastic lymphomas, a polycystic kidney phenotype and renal cell carcinoma derived from tubular epithelial cells, which are both tissues that had so far not been known to express UG. Immunohistological studies in UG/MYC transgenics and in a transgenic line (UG/eGFP) expressing Green Fluorescent Protein confirmed that the uteroglobin promoter is not only active in Clara cells, but also in tubular epithelial cells of the kidney and in lymphatic tissue. The UG/MYC transgenics will be useful to investigate the biochemical mechanisms underlying the development of carcinomas and the oncogenic properties of c-MYC in epithelial cells of various tissues.

Expression of synaptic vesicle trafficking proteins in the developing rat pineal gland.

Adult mammalian pinealocytes contain several synaptic membrane proteins that are probably involved in the regulation of targeting and exocytosis of synaptic-like microvesicles (SLMVs). Immunohistochemical techniques have now demonstrated the spatiotemporal expression pattern of some of these proteins during rat pineal ontogenesis. Various synaptic vesicle trafficking proteins are detectable in proliferating epithelial cells of the pineal anlage even at embryonic day 17.5 (E 17.5), with the exception of syntaxin I (weakly expressed from E 19.5) and dynamin I (whose levels increase markedly during the first postnatal week). Numerous cells exhibiting strong immunoreactivity for synaptobrevin II, SNAP-25, synaptophysin, and munc-18-1 are distributed throughout the increasingly compact gland at E 19.5 and E 20.5; however, their number declines toward the proximal deep part of the organ. Groups of postmitotic cells situated at the surface of the developing gland exhibit marked immunoreactivity for the aforementioned proteins and lie close to the laminin-immunoreactive outer limiting basement membrane or to its remnants in regions of basement membrane dissolution. We also show that synthesis of vimentin and S-antigen seems to begin earlier during pineal development than previously recognized. Thus, synaptic vesicle trafficking proteins are the earliest molecular markers of pinealocyte differentiation known to date, being expressed well before the onset of rhythmic hormone secretion in the pineal gland, where they may play a role in morphogenetic events. Components of the extracellular matrix such as laminin may be critically involved in the upregulation of synaptic membrane protein expression. The dynamin immunostaining pattern indicates that SLMVs of pinealocytes begin to undergo regulated cycles of exo/endocytosis during postnatal week 1.

Immunohistochemical study of the glutamate transporter proteins GLT-1 and GLAST in rat and gerbil pineal gland.

Several investigations performed during this decade have led to the hypothesis that small secretory vesicles of pinealocytes (generally referred to as synaptic-like microvesicles, SLMVs) are components of a system for intercellular paracrine communication between pineal cells, which shares many features with the process of synaptic neurotransmission. According to a recent study, one parallel that can be drawn to synaptic signal transduction seems to be the presence of pineal re-uptake systems for messenger molecules released from SLMVs, i.e. for neuroactive amino acids such as L-glutamate. In order to further characterize these uptake mechanisms, we have carried out an immunohistochemical study to explore the presence and cellular localization of the glutamate transporters GLT-1 and GLAST in rat and gerbil pineal glands. GLT-1 and GLAST were always detected in a subpopulation of pineal parenchymal cells in both species. Using immunostaining of serial semithin sections with antibodies against marker proteins of pineal cell types, GLT-1- and GLAST-positive cells were identified as interstitial glial cells. In addition, some pinealocytes also displayed immunoreactivity for GLT-1. In contrast to current thinking, our findings show that GLT-1 is not the only glutamate transporter subtype expressed in the pineal gland. Moreover, our observations point to a significant participation of interstitial cells in the process of pineal glutamatergic communication, reminiscent of the role of glial cells during glutamatergic neurotransmission in the central nervous system.

Interstitial glial cells of the gerbil pineal gland display immunoreactivity for the metabotropic glutamate receptors mGluR2/3 and mGluR5.

Recent studies have strengthened the hypothesis that neuroactive amino acids such as L-glutamate play an important role in the physiology of the mammalian pineal gland. In particular, there is now considerable evidence that L-glutamate is liberated from electron-lucent microvesicles of pinealocytes for a paracrine modulation of melatonin synthesis and release which may at least partially be mediated by the metabotropic glutamate receptor mGluR3. In order to expand our incomplete knowledge of possible pineal target cells and signal transduction mechanisms which are involved in glutamate-dependent intercellular communication, we have performed an immunohistochemical study of the gerbil pineal gland with antibodies directed against the metabotropic glutamate receptors mGluR2/3 and mGluR5. Using microwave irradiation of cryostat sections prior to immunostaining, strong immunoreactivity for both receptor subtypes was constantly observed in a subpopulation of pineal cells. Interestingly, these mGluR-positive cells could be identified as interstitial glial cells since they were labeled by antibodies against the intermediate filament protein vimentin in double immunofluorescence histochemistry. This indicates that interstitial glial cells in the gerbil possess the capacity to express at least two metabotropic glutamate receptors coupled to different intracellular signal transduction pathways. Therefore, it can be concluded that the glutamatergic communication system of the pineal gland may not only enable paracrine crosstalk among pinealocytes but probably also relies on interactions between pinealocytes and interstitial cells analogous to neuronal-glial signaling.

Immunoreactivity for multiple GABA transporters (GAT-1, GAT-2, GAT-3) in the gerbil pineal gland.

In order to further elucidate the role of gamma-aminobutyric acid (GABA) within the mammalian pineal gland, an immunocytochemical study was performed aimed at providing information on the occurrence and localization of the plasma-lemmal GABA transporters GAT-1-3 in the gerbil pineal organ. Whereas all three transporter subtypes were regularly present in this endocrine tissue, their cellular distribution differed. The analysis of serial semi-thin sections showed that pinealocytes as well as interstitial glial cells contain immunocytochemically detectable amounts of GAT proteins, indicating that both pineal parenchymal cell types participate in GABA reuptake. These results lend additional support to the hypothesis that GABA serves important physiological functions in the pineal gland.

Synaptic-like microvesicles in mammalian pinealocytes.

The recent deciphering of the protein composition of the synaptic vesicle membrane has led to the unexpected identification of a compartment of electron-lucent microvesicles in neuroendocrine cells which resemble neuronal synaptic vesicles in terms of molecular structure and function. These vesicles are generally referred to as synaptic-like microvesicles (SLMVs) and have been most intensively studied in pancreatic beta-cells, chromaffin cells of the adrenal medulla, and pinealocytes of the pineal gland. This chapter focuses on the present knowledge of SLMVs as now well-established constituents of mammalian pinealocytes. I review the results of morphological, immunocytochemical, and biochemical studies that were important for the characterization of this novel population of secretory vesicles in the pineal organ. The emerging concept that SLMVs serve as a device for intercellular communication within the pineal gland is outlined, and unanswered questions such as those pertaining to the physiological function and regulation of pineal SLMVs are discussed.

Munc-18-1 and cysteine string protein (csp) in pinealocytes of the gerbil pineal gland.

Mammalian pinealocytes contain several synaptic membrane proteins which probably play a role in the targeting and exocytosis of secretory vesicles, in particular of synaptic-like microvesicles (SLMVs). The latter are considered as the endocrine equivalent of neuronal synaptic vesicles. By means of immunocytochemical techniques and immunoblot analyses, we now show that two further key components of the molecular apparatus regulating neurotransmitter release are present in the gerbil pineal gland, i.e., munc-18-1 and cysteine string protein (csp). In addition to varicosities of nerve fibres, munc-18-1 and csp could be localized to pinealocytes where both proteins were markedly enriched in process swellings. When using antibodies against csp for an immunogold electron-microscopic study of pinealocytes, gold particles consistently decorated profiles of pleomorphic SLMVs. Interestingly, we found that also the cytosolic protein munc-18, which is partially recruited to the plasmalemma in neurons, was associated to a significant extent with SLMVs of pinealocytes and synaptic vesicles of neurons, respectively. This localization implies that munc-18 at least partially exerts its regulatory functions while being bound to secretory vesicle membranes. Our results indicate that in endocrine cells such as pinealocytes the synaptic proteins munc-18-1 and csp play essential roles during the life cycle of SLMVs.

Developmental pattern of cell type-specific calretinin immunoreactivity in the postnatal gerbil pineal gland.

The postnatal development of immunoreactivity for the neuronal calcium-binding protein calretinin in the pineal gland of the Mongolian gerbil was investigated using immunostaining of serial semithin sections. Calretinin-positive pineal cells could readily be visualized from the day of birth (P0) onwards and coexpressed the intermediate filament (IF) protein vimentin. During the first half of the first postnatal week, many of the calretinin-/vimentin-positive cells were also immunopositive for synaptophysin and neuron-specific enolase (NSE) and thus corresponded to pinealocytes. However, the expression of calretinin in pinealocytes was only transitory and declined towards the end of the first postnatal week. Thereafter, calretinin immunoreactivity became restricted to vimentin-positive interstitial glial cells. Therefore, in the gerbil pineal gland, calretinin obviously is not required in mature pinealocytes but instead serves as yet unknown functions in interstitial cells. The unusual calretinin expression pattern adds to the notion that pineal interstitial cells differ from glial cells of other brain regions. This conclusion is also underlined by our present detection of the neuronal marker protein PGP 9.5 in interstitial cells during postnatal development.

Developmental pattern of cell type-specific calretinin immunoreactivity in the postnatal gerbil pineal gland

The postnatal development of immunoreactivity for the neuronal calcium-binding protein calretinin in the pineal gland of the Mongolian gerbil was investigated using immunostaining of serial semithin sections. Calretinin-positive pineal cells could readily be visualized from the day of birth (P0) onwards and coexpressed the intermediate filament (IF) protein vimentin. During the first half of the first postnatal week, many of the calretinin-/vimentin-positive cells were also immunopositive for synaptophysin and neuron-specific enolase (NSE) and thus corresponded to pinealocytes. However, the expression of calretinin in pinealocytes was only transitory and declined towards the end of the first postnatal week. Thereafter, calretinin immunoreactivity became restricted to vimentin-positive interstitial glial cells. Therefore, in the gerbil pineal gland, calretinin obviously is not required in mature pinealocytes but instead serves as yet unknown functions in interstitial cells. The unusual calretinin expression pattern adds to the notion that pineal interstitial cells differ from glial cells of other brain regions. This conclusion is also underlined by our present detection of the neuronal marker protein PGP 9.5 in interstitial cells during postnatal development.

Rodent pancreatic islet cells contain the calcium-binding proteins calcineurin and calretinin.

Calcium is known to be of critical importance for hormone secretion in the insulin-producing B-cells of the endocrine, pancreas. Calcium-mediated intracellular signal transduction and the regulation of the concentration of free calcium in B-cells probably involve calcium-binding proteins. In the present study, we have investigated the expression of the calcium/calmodulin-dependent phosphatase, calcineurin, and the EF-hand calcium-binding protein, calretinin, in pancreata of hamsters, gerbils, and rats by immunocytochemistry. Immunocytochemical investigations of serial semithin sections of plastic-embedded pancreata revealed that calcineurin and calretinin were constantly present in islet cells of all three species. In addition to B-cells, these proteins could also be detected in glucagon (A-), somatostatin (D-), and pancreatic polypeptide (PP-) cells. Non-B-cells, especially glucagon-producing A-cells, often exhibited a significantly higher degree of immunoreactivity for both calcium-binding proteins than B-cells. Thus, calcineurin and calretinin may play distinct roles in the regulation of calcium-dependent secretory activities of the different pancreatic endocrine cell types.

Expression of synaptic membrane proteins in gerbil pinealocytes in primary culture.

Pinealocytes of various mammalian species contain abundant synaptic-like microvesicles (SLMVs) which are considered the endocrine equivalent of neuronal synaptic vesicles. Although the pinealocytes may thus be a suitable cellular model for experimental in vitro studies of SLMVs, nothing is known about the presence of SLMVs in isolated pinealocytes maintained under tissue culture conditions. In the present investigation, we prepared dissociated primary cultures of gerbil pinealocytes to study the expression and distribution of protein components of synaptic vesicles/SLMVs and the presynaptic plasmalemma in pinealocytes kept in vitro. Using immunofluorescence microscopy, we found that cultured pinealocytes readily expressed all synaptic membrane proteins investigated, i.e., synaptophysin, synaptotagmin I, synaptobrevin II, syntaxin I and SNAP-25. Punctuate immunoreactivity for the vesicle-associated proteins could be detected throughout the cell bodies of pinealocytes and was also distributed into all of their processes which began to develop within the first days in culture. Outgrowing processes exhibited growth cone-like structures which were enriched in synaptic vesicle-associated proteins. After 1 week in vitro, pinealocytes had frequently formed an elaborate network of long interwoven processes. Accumulations of synaptic vesicle-associated proteins were observed in varicosities and terminal swellings of the processes. The vesicle-rich process swellings often established synaptic-like process swellings often established synaptic-like contacts with somata and processes of other pinealocytes. Some of the pinealocyte processes possessed additional axon-like properties as demonstrated by their lack of immunoreactivity for the somato-dendritic marker MAP2 and the transferrin receptor. The comparison of the staining patterns for synaptophysin and the endocytotic marker transferrin receptor by confocal laser scanning microscopy revealed a largely differential intracellular distribution of the two proteins. This may indicate that a substantial fraction of pinealocyte SLMVs by-passes the early endosomal-related recycling pathway of SLMVs. Herewith, we have shown that isolated gerbil pinealocytes maintained in primary culture can acquire morphological and neurochemical traits which closely mimick those observed in vivo. In particular, these cultures permit experimental studies of the compartment of pinealocyte SLMVs which seem to make up a major secretory pathway for paracrine intrapineal communication.

Rat and gerbil pinealocytes contain the synaptosomal-associated protein 25 (SNAP-25).

It has recently been established that the neuroendocrine pinealocytes of mammals contain several synaptic membrane proteins that are involved in the regulation of vesicle trafficking in the nerve terminal. In the present study, we have conducted immunoblot and immunocytochemical analyses to demonstrate that another key component of the presynaptic plasmalemma, i.e., protein SNAP-25 (synaptosomal-associated protein 25 kDa), can be detected in pinealocytes. Immunostaining of serial semi-thin sections of plastic-embedded rat and gerbil pineals with monoclonal SNAP-25 antibodies showed that SNAP-25 was present in pinealocytes of both species. We proved its coexpression with other synaptic membrane proteins (synaptophysin, synaptotagmin I, synaptobrevin II, and syntaxin I) at the single cell level. Thus, pinealocytes obviously are endowed with the major proteins that are thought to regulate the targeting and exocytosis of secretory vesicles, in particular of synaptic-like microvesicles.

Synaptotagmin I, synaptobrevin II, and syntaxin I are coexpressed in rat and gerbil pinealocytes.

Recent studies have shown that mammalian pinealocytes contain a compartment of synaptic-like microvesicles that may serve secretory functions; however, knowledge of the molecular composition of these microvesicles is still incomplete. Therefore, we have analyzed rat and gerbil pineal glands for the presence of synaptotagmin I, synaptobrevin I and II, syntaxin I, and synaptoporin (synaptophysin II) by immunoblot analyses and immunostaining of serial semithin sections. These proteins, which are components of the synaptic vesicle membrane or presynaptic plasmalemma, are thought to be essential for synaptic vesicle trafficking and exocytosis. Antibodies against synaptotagmin I, synaptobrevin II, and syntaxin I label pinealocytes (identified with an antiserum directed against synaptophysin I) in pineal glands of both species, the coexpression of the latter proteins being demonstrable at the single cell level. In contrast, pinealocytes are not or only weakly stained by the synaptoporin antibody. Immunoreactivity for synaptobrevin I is restricted to intrapineal nerve terminals, thus indicating a differential expression of synaptic vesicle protein isoforms within endocrine tissues. Immunogold staining has been performed in the gerbil pineal and reveals that synaptobrevin II and synaptotagmin I can be localized to the synaptic-like microvesicles that are concentrated in pinealocyte process terminals. Syntaxin immunoreactivity is associated with clear microvesicles and with the plasma membrane. Our findings corroborate the hypothesis that the synaptic-like microvesicles of pinealocytes can be considered as the endocrine equivalent of neuronal synaptic vesicles. Since pinealocytes of several mammalian species contain abundant clear microvesicles, the pinealocyte may serve as a paradigm for studies aimed at elucidating the biogenesis and functions of synaptic-like microvesicles in neuroendocrine cells.

Differential immunocytochemical localization of calretinin in the pineal gland of three mammalian species.

Calcium plays an important role for signal transduction in the mammalian pineal organ. The regulation of the intracellular concentration of free calcium probably involves calcium-binding proteins of the calmodulin superfamily. In the present study, we have investigated the expression of calretinin, one member of this superfamily, in the pineal organ of hamsters, gerbils and guinea-pigs by means of immunochemical and immunocytochemical analyses with a calretinin-specific antiserum. In immunoblots this antibody recognized a single protein band of approximately 29 kDa in the brain and pineal organ of all three mammalian species. Immunocytochemical investigations of serial semithin sections of plastic-embedded pineals revealed the constant occurrence of variable numbers of calretinin-positive cells throughout all glands. In order to identify the immunopositive cells precisely, adjacent sections were exposed to antibodies against various marker proteins of pineal cell types, i.e., synaptophysin, neuron-specific enolase, protein gene product 9.5, S-antigen, vimentin and S-100. By this approach, calretinin could be localized to vimentin-positive cells in the gerbil which are generally considered as interstitial glial cells. Likewise, calretinin-positive cells in the guinea-pig probably correspond to interstitial cells, taking into account their morphology and the lack of calretinin immunoreactivity in pinealocytes. The unusual expression of calretinin in astrocyte-like cells further supports the notion that pineal glial cells are endowed with peculiar properties. In contrast to gerbil and guinea-pig, a subpopulation of pinealocytes displayed calretinin immunoreactivity in the hamster. This finding adds to the hypothesis that in pinealocytes of some species calretinin plays a role in calcium-mediated signal transduction which eventually is linked to melatonin synthesis. Our results demonstrate that calretinin is a regular constituent of pineal glands in three mammalian species, but that its cellular localisation shows interspecific variation. This variation suggests that the protein is involved in diverse calcium-mediated functions in the mammalian pineal gland.

Differential distribution of synaptotagmin I and rab3 in the anterior pituitary of four mammalian species.

In the anterior pituitary of rat, gerbil, hamster and guinea pig, the presence and cellular distribution of the synaptic vesicle-associated proteins synaptotagmin I and rab3 were analyzed by immunoblotting and by immunocytochemical staining of serial semithin sections. Our results show that rab3 proteins are ubiquitously expressed in all endocrine cell types of both the anterior and intermediate lobe. In many cells, rab3 immunoreactivity was concentrated beneath the plasmalemma. This intracellular distribution coincided with the distribution of secretory granules, suggesting a possible association of rab3 proteins with the latter organelles. The staining patterns observed using two monoclonal rab3 antibodies with different isoform specificities are compatible with the recent suggestion that rab3B is the dominant rab3 isoform in anterior pituitary cells. However, we could demonstrate that also rab3A is present in endocrine adenohypophyseal cells, albeit at low levels. In contrast to rab3, synaptotagmin I immunoreactivity was only detected in a limited number of adenohypophyseal endocrine cells. Whereas the monoclonal synaptotagmin I antibody consistently failed to immunostain lactotrophs and endocrine cells of the intermediate lobe, other endocrine cell types displayed variable immunoreactivities towards this antibody. Although a low level of synaptotagmin I expression in the immunonegative cells cannot be excluded, the above observation may reflect a differential distribution of synaptotagmin isoforms in endocrine organs, as it has been described for the nervous system. Our study has established that endocrine cells of the anterior pituitary are endowed with proteins of the rab3 and synaptotagmin families which are generally thought to play important roles in the regulation of the trafficking and/or exocytosis of secretory organelles and, hence, probably fulfil similar functions in adenohypophyseal cells.

Bronchiolar nonciliated secretory (Clara) cells: source of guanylin in the mammalian lung.

The peptide guanylin, which has recently been isolated from the intestine, is involved in the regulation of fluid secretion in the intestinal epithelium by activation of guanylate cyclase C, the putative guanylin receptor. Since the latter protein is also expressed in airway epithelia, we investigated the lung of three mammalian species for the presence and cellular localization of guanylin by immunoblot (Western blot) analyses and light and electron microscopical immunocytochemistry. In Western blots of bovine, guinea pig, and rat lung extracts, three different guanylin antisera directed against the midportion and against the C terminus of the precursor molecule identified a peptide band corresponding to the apparent molecular mass of guanylin. Localization studies in the lung revealed that guanylin is exclusively confined to nonciliated secretory (Clara) cells in the lining of distal conducting airways. The presence of guanylin in the lung and particularly its specific localization to Clara cells indicate that these cells may play a pivotal role in the local (paracrine) regulation of electrolyte/water transport in airway epithelia.

The ras-like rab3A protein is present in pinealocytes of the gerbil pineal gland.

As part of our molecular and functional characterization of the compartment of synaptic-like microvesicles (SLMVs) in mammalian pinealocytes, we have now analyzed the pineal gland of the Mongolian gerbil for the presence of rab3 proteins. Members of this subfamily of small G proteins are thought to regulate the intracellular trafficking and/or membrane fusion of secretory vesicles. Immunostaining of serial semi-thin sections of plastic-embedded pineals with monoclonal antibodies which recognize rab3A revealed the occurrence of rab3A in pinealocytes throughout the gland. Rab3A immunoreactivity was markedly enriched in dilated pinealocyte process terminals known to contain accumulations of SLMVs. The latter could be labeled with the rab3 antibodies at the ultrastructural level when immunogold staining was performed. Our results lend further support to the hypothesis that mammalian pinealocytes are endowed with a population of SLMVs that serve secretory functions.