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. Author manuscript; available in PMC: 2021 Mar 30.
Published in final edited form as: J Neurochem. 2019 Oct 20;151(5):558–569. doi: 10.1111/jnc.14846

Gap junctions coordinate the propagation of glycogenolysis induced by norepinephrine in the pineal gland

Eliseo A Eugenin 1,*, Silvana Valdebenito 1, Anna Maria Gorska 1, Agustin D Martínez 2, Marcela Bitran 3, Juan C Sáez 2,3,*
PMCID: PMC8009014  NIHMSID: NIHMS1044818  PMID: 31381153

Abstract

Chemical and electrical synapses are the two major communication systems that permit cell-to-cell communication within the nervous system. Although most studies are focused on chemical synapses (glutamate, GABA, and other neurotransmitters), clearly both types of synapses interact and cooperate to allow the coordination of several cell functions within the nervous system. The pineal gland has limited independent axonal innervation and not every cell has access to nerve terminals. Thus, additional communication systems, such as gap junctions, have been postulated to coordinate metabolism and signaling. Using acutely isolated glands and dissociated cells, we found that gap junction spread glycogenolytic signals from cells containing adrenoreceptors to the entire gland lacking these receptors. Our data using glycogen and lactate quantification, electrical stimulation, and HPLC-EC, demonstrate that gap junctional communication between cells of the rat pineal gland allows cell-to-cell propagation of norepinephrine (NE)-induced signal that promotes glycogenolysis throughout the entire gland. Thus, the interplay of both synapses is essential for coordinating glycogen metabolism and lactate production in the pineal gland.

Keywords: Glycogen, connexin, electrical and chemical synapses, hemichannels

In this issue:

graphic file with name nihms-1044818-f0001.jpg

Gap junctions play a key role in the metabolic and electrical coordination of several tissues. Here, we demonstrate that gap junctions between astrocytes and neurons, pinealocytes, are essential to spread glycogen degradation from cells containing adrenergic receptors into cells without innervation. Thus, gap junctions are essential to coordinate the metabolism of the entire gland.

Introduction

The rat pineal gland is a neuroendocrine organ innervated by sympathetic neurons that end in the center of the gland, coordinating the light/dark cycle for the production of melatonin. The activity of the pineal gland is driven by the suprachiasmatic nucleus, which regulates the phasic release of norepinephrine (NE) from sympathetic neurons (Colwell 2000b, Colwell 2000a). NE action is transduced post-synaptically by β- and α1- adrenoreceptors (Axelrod 1974, Klein et al. 1981, Eugenin et al. 1997), through which it induces synthesis and releases of melatonin, a hormone that is involved in the photoperiodic regulation of reproduction and several other physiologic functions (Reiter 1980).

The rat pineal gland is made up mainly by pinealocytes, which are responsible for melatonin synthesis, and astrocytes (Freund et al. 1977), which constitute around 5–10% of the total cell content in the gland (Moller 1978, Moller et al. 1978, Schachner et al. 1984). A key source of energy within the CNS is glycogen, which is mainly present in astrocytes. Glycogen stores are dynamic and are reduced during the dark periods (Kachi & Ito 1977), suggesting that sympathetic activity induces glycogen degradation. Our published data indicate that in the rat pineal gland, glycogen stores are localized mainly in astrocytes and less in pinealocytes (Eugenin et al. 1997). NE induces a rapid reduction in the amount of pineal glycogen through activation of α1- and β- adrenoreceptors (Eugenin et al. 1997). However, the mechanism of transmission of glycogenolytic signals into the entire gland is unknown.

Gap junctions (GJs) are clusters of intercellular channels permeable to ions and small molecules, including second messengers such as cyclic nucleotides (Pitts & Simms 1977, Malik & Eugenin 2017, Valdebenito et al. 2018, Eugenin 2014), Ca2+, and IP3 (Saez et al. 1989), thereby allowing electrical and metabolic synchronization among coupled cells (Stauffer et al. 1993, Combettes et al. 1994, Eugenin et al. 1998, Dupont et al. 2000a, Dupont et al. 2000b, Nathanson et al. 1999b, Burgstahler & Nathanson 1998). At least 21 connexin (Cx) genes have been identified in the human genome. The same Cx type can be expressed in different cells, and more than one Cx can be expressed by the same cell (Willecke et al. 2002). In the pineal gland, not every pinealocyte receives independent innervation (Vollrath et al. 1985, Vollrath 1985, Ichimura et al. 1986). It has been proposed that intercellular communication through GJ among pineal cells could permit non-innervated cells to respond to neurotransmitters (Saez et al. 1991). Various studies have demonstrated the presence of GJs between pinealocytes (Wolfe 1965, Taugner et al. 1981, Saez et al. 1991), between astrocytes (Saez et al. 1991, Berthoud & Saez 1993) and between pinealocytes and astrocytes (Cieciura & Krakowski 1991). In the rat pineal gland, Cx43 and Cx26 have been identified in GJs formed between astrocytes and pinealocytes, respectively (Saez et al. 1991), but also Cx36 has been identified in pinealocytes (Wang et al. 2017, Belluardo et al. 2000). Nonetheless, the metabolic role of the functional coupling between pinealocytes and astrocytes have not been reported.

In this report, we show that gap junctional communication is essential to coordinate and amplify glycogenolytic signals generated in sympathetic neurons into areas lacking direct neuronal innervation.

Materials and Methods

Reagents.

Norepinephrine (NE) (Cat# A7257), rabbit liver glycogen (Cat# G8876), octanol (Cat# 297887), 18α-glycyrrhetinic acid (Cat# G8503), acetonitrile (Cat# 100030), quinine (Cat# 69311), dihydroxybenzylamine (Cat# 858781), sodium octyl sulfate (Cat# O4003), sulfuric acid (Cat# 339741) and a monoclonal anti-GFAP (RRID:AB_477010) antibody were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Trypsin (Cat# 06369880103) and collagenase (Cat# 05349907103) were obtained from Roche CustomBiotech (Indianapolis, IN, USA). DME/F12 medium (Cat# 11320082) (1:1) and fetal bovine serum (FBS) (Cat# 16000044) were obtained from ThermoFisher (Grand Island, NY, USA). Phenol (Cat# 33517) was obtained from Sigma Chemical Co. (St. Louis, MO, USA). Goat anti-mouse IgG antibody conjugated to Cy5 (AlexaFluor 647) (RRID: AB_2535804) and donkey anti-rabbit IgG antibody conjugated to FITC (AlexaFluor 488) (RRID: AB_2535792) was obtained from ThermoFisher (Grand Island, NY, USA). Blocking peptides for Cx43 were synthetized in Genscript (Piscataway, NJ). Cx43 blocking peptide, Gap26, VCYDKSFPISHVR, and Scrambled peptide, PSFDSRHCIVKYV were used.

Animals source.

Sprague-Dawley rats (RRID: RGD_10395233) weighing ~180–200 g (PND= ~60, 8 weeks) of both genders were obtained from the Animal Institute of the Pontificia Universidad Católica de Chile. All animal experiments were compliant with the animal ethics requirements of the Pontificia Universidad Católica de Chile. Certification of Animal Protocol Approval was provided by the Ethics Committee of the Pontificia Universidad Católica de Chile (Protocol: PUCFis2000). The animals were housed in groups of 4 in a transparent polypropylene cage, 22” long × 12.5” wide × 8” deep, in a room with controlled temperature and humidity under a 12 h light and 12 h dark cycle with constant access to water and food. During the last 2 h of the light period, rats were decapitated without anesthesia, because most anesthetic blocks calcium and GJ channels (Zhang et al. 2016, Miyata et al. 2015, Voss et al. 2014, Del Re & Woodward 2005), and the pineal glands were carefully removed, and placed in cold phosphate-buffered saline (PBS). Special care was taken not to disrupt the leptomeninges that surrounds the gland. Our animal experiments were not performed blindly. However, all analyses of samples obtained from whole glands and tissue cultures were performed blinded.

Acute organotypic culture and drug treatments.

Freshly dissected pineal glands were cultured as described (Eugenin et al. 1997). Rats were decapitated without anesthesia, and pineal glands were collected in cold PBS and using a razor blade under a dissecting microscope, a small cut was applied to each lobule to disrupt the leptomeninges and allow the access of the drugs to the pineal cells. As a control, glands without leptomeninges disruption were used. Gap junction blockers, octanol (O) and 18α-glycyrrhetinic acid (AGA), were added 2 – 3 min before the application of NE. Stock solutions of octanol (5% in ethanol) and 18α-glycyrrhetinic acid (x500 in DMSO) were prepared, and dilutions of each were done in culture medium right before their use. Controls with the vehicle, ethanol, and DMSO did not induce nonspecific effects (data not shown).

Measurement of glycogen.

The total amount of glandś glycogen was measured using a previously reported method (Lo et al. 1970) with minor modifications as described (Eugenin et al. 1997). Three intact or 15 dissociated pineal glands were utilized to measure the detectable amount of glycogen. Glycogen concentrations were obtained from standard curves generated with stock glycogen solutions of known concentration (Eugenin et al. 1997). Also, pineal glands without leptomeningeal disruption did not show significant glycogen degradation even in the presence of NE, indicating that leptomeninges was intact and that NE only acted on the cells exposed in the sectioned area (data not shown). Results were calculated as μg of glycogen/mg of protein and then normalized to control glands to obtain the percentage of reduction in glycogen content. The protein concentration of the sample was determined using the Lowry method (Lowry et al. 1951).

Dissociated pineal cells.

Cells were dissociated as described previously (Saez et al. 1991). Rats were decapitated without anesthesia, and fifteen pineal glands were dissected, and placed in D-MEM/F12 medium containing penicillin (5000 U/ml) and streptomycin (5 mg/ml) (Cat# 15070063) (ThermoFisher, Grand Island, NY, USA) and cut into 8–10 pieces under a dissecting microscope. Then, gland fragments were dissociated with 0.2% trypsin and 0.06% collagenase for 45 min at 37°C in a tissue culture incubator (5% CO2, 95% air and 100% humidity atmosphere). Cells were disaggregated by gentle trituration through a glass Pasteur pipette, centrifuged at 1,000 r.p.m. for 1 min in a bench top centrifuge, washed twice with sterile D-MEM/F12 supplemented with 10% FBS and incubated in the same medium for 15 min at 37°C in a tissue culture incubator under the same atmosphere described above. Sub-confluent and confluent cells were then transferred to 30 mm diameter plastic Petri dishes and used for glycogen degradation experiments. After treatment with different agents, the cells were centrifuged, and the pellet was processed for glycogen determination, and the medium was collected for lactate determination.

Transmural stimulation of rat pineal glands.

Rats were euthanized by decapitation without anesthesia to prevent the unspecific effects of anesthetic in calcium and GJ channels. Six to eight glands per experiment were transferred to 800 μl chambers equipped with platinum electrodes and superfused with a Krebs-Hepes buffer (in mM: 120 NaCl (Cat# S7653), 5 KCl (Cat# P9333), 2.6 CaCl2 (Cat# C1016), 0.67 MgSO4 (Cat# 203726), 0.03 EDTA (Cat# EDS, CAS# 60-00-4), 0.06 ascorbic acid (Cat# A5960), 11 glucose (Cat# G5767) and 20 mM Hepes (Cat# H3375), pH 7.4) (Sigma Chemical Co., St. Louis, MO, USA). The latter solution was supplemented with 1 μM yohimbine (Cat# Y3125) and 2 μM desipramine (Cat# D3900) (Sigma Chemical Co., St. Louis, MO, USA) to prevent NE reuptake and kept at 37°C under continuous bubbling with 95% O2 and 5% CO2. The protocol consisted of three periods of electrical stimulation, in which the first (S1) served as an internal control to evaluate the amount of NE released. The effects of octanol or 18α-glycyrrhetinic acid on the glycogen content and release of endogenous NE were assessed after the second stimulation (S2), while the recovery effect was evaluated after the third stimulation (S3). Each stimulation period consisted of a train of square electrical pulses (5 Hz, 1 ms) delivered for 1 min every other minute over 10 min (1,500 pulses per stimulation). The pineal glands were superfused at a flow rate of 1.0 ml/min, except during stimulation and the corresponding baseline collection periods (B1 and B2). The solution contained in the chambers during the basal and stimulation periods was immediately collected in tubes containing 35 μl of perchloric acid (Cat# 244252), and 5 μl of 5% Na2S2O3 (Cat# 72049) (Sigma Chemical Co., St. Louis, MO, USA) and immediately processed for HPLC determination of NE concentration.

Electrochemical quantification of endogenous norepinephrine (eNE) release from pineal glands.

NE was measured by HPLC-EC, as described (Torres et al. 1992). Supernatants collected during the basal and stimulation protocols were processed by adsorption chromatography in alumina (Anton & Sayre 1962) before their injection into, the HPLC system. Dihydroxybenzylamine (21 pmol/sample) was added as an internal standard. NE and dihydroxybenzylamine were eluted from the alumina with 300 μl 0.1 N perchloric acid. The samples were injected onto a lichrosphere reverse-phase ion-pair column (60 RP Select B, Merck 50981, 5 μm, 124 × 4 mm). The mobile phase (in mM: 100 NaH2PO4 (Cat# S8282), 0.9 Na2EDTA (Cat# 324503), 1.15 sodium octyl sulfate and acetonitrile, pH 2.7) (Sigma Chemical Co., St. Louis, MO, USA) flowed at a rate of 1.0 ml/min. The electrochemical detection system (656 ED, Metrohm) consisted of a glassy carbon working electrode and an Ag/AgCl reference electrode set at a potential of 0.8 V with an amperometric detector (641 VA, Metrohm) connected to a Merck Hitachi D-2500 integrator.

Histochemistry.

Pineal glands were washed in PBS, fixed in Bouin, embedded in methacrylate and semi-thin sections (1 μm) were then obtained. Glycogen was detected with periodic acid-Schiff (PAS) reagent, as was described previously (Eugenin et al. 1997).

Evaluation of dye coupling in whole pineal glands.

To evaluate the degree of cell-to-cell communication in the whole glands, we determined the diffusion of 0.05% Rhodamine-B (Cat# 83689) or Dextran-FITC (Cat# 46945) (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) from cells at the side, where the leptomeninges was disrupted, in close contact with a thin layer of the dye for 5 min at room temperature. Then, the dye solution was discarded, and the glands were rinsed with PBS. Control plates were set by exposing the cells under similar conditions to the dye mixture but without scraping. The cell types loaded with Rhodamine-B were identified after the diffusion time, and the distance of the dye diffusion was quantified.

Dye coupling between cultured cells.

Gap junctional communication was tested by observing the intercellular transfer of Lucifer Yellow (5% w/v LY dissolved in 150 mM LiCl) microinjected through glass microelectrodes by brief overcompensation of the negative capacitance circuit in the amplifier until the impaled cell was brightly fluorescent. After dye injection, cells were observed for 1–4 min to determine whether dye transfer occurred as described previously (Eugenin et al. 1998). Dye coupling between cultured cells was observed in an inverted Z1 microscope with a redundant incubation system (2 redundant incubation systems) to assure minimal changes in temperature (Zeiss, Germany).

Lactic acid quantification.

Lactate release was evaluated using a lactate assay kit (Sigma, MA. Cat#MAK064), according to the manufacturer’s instructions. The medium from whole pineal gland was mixed with 4 volumes of lactate assay buffer and centrifuge at 13,000 g for 10 minutes. Master reaction mix contains 20 μl sample solution, 26 μl lactate assay buffer, 2 μl lactate enzyme mix, and 2 μl lactate probe. Sample absorbance was measured at 570 nm (A570) on a microplate reader. A total of 10 μl of 100 nmole/μl lactate standard was diluted with 990 μl lactate assay buffer to generate a 1nmole/μl standard solution. The volumes of the 1 nmole/μl lactate standard solution were used to generate the standard curve of 0, 2, 4, 6, 8, and 10 nmole.

Data acquisition.

No data points were eliminated, and no sample size was pre-calculated. However, according to our previous data and publication, the size selected was sufficient (Eugenin et al. 1997). Our study was not a pre-register study according to NIH regulations.

Statistical Analysis.

Mean non-parametric Kruskal-Wallis analysis was used to test for differences in percentage. If a significant F-value was obtained, means were compared with Bonferroni-Dunn multiple comparison tests. A value of *p<0.005 as compared to control conditions was considered significant. A value of #p<0.007 as compared to NE conditions was considered significant. Multiple comparisons give similar results. For all graphs, each n corresponds to 4 to 7 independent measures to assure proper determinations. To perform these analyses, Origin 8 and GraphPad Prism 7.0 were used.

Results

Norepinephrine-induced glycogenolytic signals that are propagated through GJ.

In the rat pineal gland, approximately 90 – 95% of the cells are pinealocytes, and about 5–10% are astrocytes (Moller et al. 1978). Previously, we demonstrated that glycogen stores are mainly localized in astrocytes, and they are sensitive to degradation upon NE application (Eugenin et al. 1997). Glycogen degradation induced by NE was mediated primarily by the activation of α1-adrenoreceptor, but not α2-, and β-adrenoreceptors (Eugenin et al. 1997). Nonetheless, activation of the β-adrenoreceptor-dependent pathway modulated the response induced by the activation of α1-adrenoreceptors, suggesting an active cross-talk between these receptors (Eugenin et al. 1997). However, the mechanism by which glycogenolytic signals generated in a few neuronal terminals are spread into the entire gland was unknown.

To study the role of GJ during the process of NE-induced glycogen degradation in the rat pineal gland, we used two different GJ blockers: octanol (O) and 18α-glycyrrhetinic acid (AGA) (Tordjmann et al. 1996, Eugenin et al. 1998). These two agents were separately pre-applied to pineal glands to test the role of gap junctional communication in NE-induced glycogenolytic response. As per our previous publication (Eugenin et al. 1997), PAS (periodic acid-Schiff) staining to identify glycogen stores under control conditions was stronger in astrocytic cells and lighter in pinealocytes (Fig. 1A). Careful isolation of the entire pineal gland resulted in an organ system fully isolated from the external stimuli due to the gland being entirely covered in the leptomeninges. Thus, the external application of NE (10−4 M) to the medium containing freshly isolated pineal glands did not result in alterations in glycogen content due to the presence of the leptomeninges (data not shown). However, if two small cuts in each lobule of the gland were performed to disrupt the leptomeninges and allow exposure of pineal cells to NE, glycogen stores were depleted in response to NE (Fig. 1, cut, cartoon). Exposure to NE for 15 min resulted in maximal glycogen degradation, even though only cells in the incisions were exposed to NE (Fig. 1A – C,~ 60%). Quantification of glycogen by PAS staining also determined that NE treatment resulted in 57 ± 8% degradation of glycogen content (Fig. 1D). Quantification of the glycogen content in the presence of GJ blockers and subsequent treatment with NE indicates that GJs are required for the transmission of the glycogenolytic effects induced by NE treatment (Fig. 1C and D, O or AGA). The application of GJ blockers alone showed similar glycogen staining to control glands (Fig. 1D, O or AGA). The prevention of the glycogen degradation in response to NE in the presence of GJ blockers was reversible because washout (W) of the blocking agents resulted in the partial recovery of the glycogenolytic function in response to new added NE (Fig. 1D, O/W, and AGA/W). These results suggest that GJs propagate glycogenolytic signals from cells in contact with NE to distal cells within the gland.

Figure 1: NE induces a reduction of glycogen content in rat pineal gland by a GJ-dependent mechanism.

Figure 1:

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Treatment of isolated rat pineal glands with 10−4 M NE for 15 min induced a dramatic reduction of glycogen stores as demonstrated by PAS staining. Under control conditions, astrocytes are more positive for glycogen staining across the entire pineal gland, G (red staining, A). NE treatment for 15 min induced a drastic decrease in glycogen staining across the entire pineal gland, G (B). The preincubation of the glands with the GJ blockers, octanol (O, 500 μM) or 18α-glycyrrhetinic acid (AGA, 32 μM), abolished the glycogenolytic effects of NE, maintaining the glycogen stores (C, corresponds to an example of AGA plus NE). G corresponds to the gland area delimited by a segmented black line. (D) Quantitative determinations of glycogen confirmed the histochemistry data in rat pineal glands in different conditions, such as NE, NE plus GJ blocker, or GJ blocker alone. Octanol (O; 500 μM) or AGA (32 μM) were used as GJ blockers. Washout of the blocker was also performed to examine reversibility (O/W or AGA/W). As a control, octanol (O) or AGA alone was also examined. Each bar corresponds to the average ± S.D. The * represents significant difference to NE treatment (*p∠0.005; n=12 of experiments using different animal sets, 105 animals were used for the analysis). The # represents significant differences between NE plus GJ blocker and washout blocker (#p∠0.005, n=12 of experiments using different animal sets, 105 animals were used for the analysis).

Gap junction blockers prevent glycogenolysis in the whole gland, but not in dissociated cells lacking gap junctional communication.

To examine whether octanol (O) and 18α-glycyrrhetinic acid (AGA) could alter glycogenolysis in a GJ-independent manner. We dissociated rat pineal glands to generate sub-confluent cultures of mixed cells to prevent cell-to-cell contact and efficient formation of GJs. Then, sub-confluent cell cultures of dissociated glands were stimulated for 15 min with NE in the presence or absence of O or AGA (Fig. 2A). NE treatment of dissociated pineal gland cells for 15 min resulted in ~25% degradation of glycogen stores (Fig. 2A, NE). The pretreatment of the cultures with octanol or 18α-glycyrrhetinic acid before NE (O+NE or AGA+NE) treatment did not affect glycogen degradation as compared to NE treatment (Fig. 2A, O+NE or AGA+NE). Treatment of the cultures with O or G alone did not prevent glycogen content (Fig. 2A, O or AGA, * as compared to NE treatment).

Figure 2: Gap junction blockers did not affect the glycogenolysis induced by NE in disaggregated pineal cells.

Figure 2:

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(A) To examine whether GJ blockers affect the transduction signals of NE receptors in the absence of functional GJ, we used dissociated pineal cells into sub-confluent cultures to evaluate the NE and GJ effect on the glycogen content of these cells. NE (10–4 M) treatment induced glycogen degradation in dissociated cells. Glycogen degradation induced by NE was not altered by the pre-application of octanol (O+NE) or 18α-glycyrrhetinic acid (AGA+NE). The addition of GJ blockers alone did not affect the basal levels of glycogen degradation (O or AGA). (B) Analysis of NE-induced glycogen degradation in confluent cultures of rat pineal glands. NE treatment-induced glycogen degradation in a GJ dependent manner. The preincubation of the glands in control or NE conditions with octanol (O) or AGA, totally abolished glycogen degradation induced by NE. Plating of the cells in the presence of the Cx43 blocking peptide (Cx43p, 300 μM) or quinine (Qui, 200 μM, a Cx36 blocker) resulting in the prevention of glycogen degradation in response to NE in a Cx43, but not Cx36, dependent manner. Scrambled (Scr) peptide did not affect NE induced glycogen degradation. Each bar corresponds to the average ± S.D. The * represents a significant difference compared to NE treatment (*p∠0.004; n=5–7 experiments using different animal sets of experiments, 126 animals were used for the analysis).

To examine the role of GJ in dissociated cells, confluent cultures of dissociated rat pineal cells were generated. Treatment of confluent cultures with NE (10−4 M) induced glycogen degradation (~60%, Fig. 2B). Pretreatment of the confluent cultures with the GJ blockers, O or AGA, for 5 min and subsequent NE addition blocked NE-induced glycogen degradation (Fig. 2B, O+NE, O, AGA+NE or AGA alone). Untreated cultures or cultures with O or AGA alone did not show any glycogen degradation under basal conditions (Fig. 2B, O or AGA). Plating the cells in the presence of Cx43 blocking peptide (Cx43p, gap26, 300 μM) resulted in significant prevention of glycogen degradation in response to NE. However, quinine (a Cx36 blocker, 200 μM) was ineffective in reducing glycogen degradation in response to NE treatment. Scrambled peptide (scr) did not affect the NE-induced degradation of glycogen stores. Thus, Cx43 is essential for the spread of glycogenolytic signals across the pineal gland. Also, these results indicate that the complementation of chemical (NE and their receptors) and GJ is required for the efficient glycogen degradation across the pineal gland.

The release of endogenous NE (eNE) by transmural electrical stimulation results in glycogen degradation in a GJ-dependent manner.

Here, we isolated fresh pineal gland, without leptomeningeal disruption, and subjected it to intramural electrical stimulation to determine whether the release of endogenous NE (eNE) could induce the degradation of glycogen. Our protocol consisted of three sequential stimulations (S1 to S3), as described in Materials and Methods, to determine the release of eNE and glycogen content.

Overall, no release of NE was detected during the entire duration of the experiment if no electrical stimulation was supplied (basal conditions, B1 to B3). Electrical stimulation-induced a consistent and reproducible release of eNE into the bath (1 to 3 pmol per bath) (Fig. 3 A and B). S1 was used to determine the basal levels of eNE release and glycogen degradation. S2 was used to examine the effects of treatments with GJ blockers, and S3 to observe the reversibility or recovery of the system (Fig. 3 A and B). These results indicated that successive stimuli did not desensitize or deplete the eNE content in the pineal sympathetic terminals under stimulatory conditions (see Fig. 3 A and B).

Figure 3: Release of endogenous NE (eNE) by electrical stimulus induces glycogen degradation by a GJ-dependent mechanism.

Figure 3:

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We examined whether endogenous NE released via an electrical stimulus-induced glycogen degradation, and the effect of GJ blockers, octanol (500 μM) and 18α-glycyrrhetinic acid (32 μM). The three identical electrical stimuli induced a release of 1 to 3 pmoles of NE each time (Graph A and B, white bars). The first stimulus was to establish basal conditions. The second stimulus was designed to apply the pharmacological agents, and the third stimulus was to examine the recovery of the system. The addition of octanol during S2 reduced the release of eNE (graph A, black bar) and reduced the glycogen degradation (graph C, black bar). 18α-glycyrrhetinic acid, AGA, did not affect the release of eNE induced by the electrical stimulus (graph B, black bar). Each bar corresponds to the average ± S.D.), but reduced the degradation of glycogen induced by eNE (graph D, black bar, each bar corresponds to the average ± S.D.). The * represents a significant difference between basal conditions (B), without the electrical stimulus and with the electrical stimulus (S) (*p≤0.005; n=6–8 of experiments using different animal sets, 420 animals were used for the analysis). The # represents significant differences between electrical stimulus without GJ blocker and with the respective GJ blocker (p≤0.005, n=7 of experiments using different animal sets, 420 animals were used for the analysis).

Octanol, but not 18α-glycyrrhetinic acid, reduces the release of eNE induced by transmural electrical stimulation.

To demonstrate that GJs are necessary for the spread of the glycogenolytic signals generated by NE across the whole gland, we applied GJ blockers during S2 and examined their effects on the amount of released eNE and gland glycogen content. During S1, we determined the baseline secretion of eNE and glycogen degradation. During S2, we applied octanol (500 μM O) or 18α-glycyrrhetinic acid (32 μM, AGA), before the electrical stimulation and S3, was designed to examine recovery of the system. Our system consisted of three periods of transmural stimulation (S1 to S3) induced a similar release of eNE to the extracellular media (1 to 3 pmol), and we did not detect eNE during the periods of non-stimulation, the basal conditions (B1 to B3). During the S2 period, octanol, but not 18α-glycyrrhetinic acid, reduced the release of eNE (Fig. 3A and B) in a reversible manner (Fig. 3A, and B). No changes in the basal release of eNE were detected (B1 to B3).

Blocking gap junctional communication prevents glycogen degradation in response to NE treatment.

Using the same pineal glands used above, quantification of glycogen content was performed at different time points post transmural electrical stimulation, B1 to B3 and S1 to S3. Electrical stimulation-induced consistent glycogen degradation (20–30%) (Fig. 3 C and D, S2). In S2, octanol (O) reduced glycogen degradation (Fig. 3C), but as described in Fig. 3 A, also significantly decreased the release of eNE. Thus, this apparent anti-glycolysis effect of octanol cannot be attributed exclusively to the GJ blockade because the amount of agonist released in response to the electrical stimuli, NE, was also reduced (compare Fig. 3 A and C). Interestingly, these effects were reversible; after octanol washout, the release of eNE and glycogen degradation were recovered during the S3 period (see Fig. 3 A and C, respectively). This paradoxical effect can be explained because octanol also blocks Ca2+ channels, which participate in the process of neurotransmitter exocytosis induced by the electrical stimuli (Tachikawa et al. 1989, Todorovic et al. 2000). In contrast, AGA did not affect the eNE release induced by electrical stimuli (see Fig. 3B, black bar) but blocked the degradation of glycogen induced by eNE (Fig. 3C, black bar), suggesting that it blocked gap junctional communication specifically and did not affect chemical transmission. In S3 the release of eNE and consistent glycogen degradation was not affected, indicating that their machinery was not affected by the transient application of this GJ blocker. These results suggest that gap junctional communication is essential for the propagation of glycogenolytic signals from cells with NE receptors to cells without the receptors within the pineal gland.

Functional GJ couples the entire pineal gland.

To determine the extent of gap junctional communication present in the gland, fresh glands were isolated with the leptomeninges surrounding them (Fig. 4). As described in Material and Methods, the glands were cut at the basal side, and this scrap region of the gland was placed in contact with a thin layer of the dye. After 15 min, in control and NE treated conditions in the presence and absence of octanol (O) or AGA, glands were fixed and sectioned for GFAP staining and the presence of the dye (Fig. 4A to D). No differences in dye diffusion (sulforhodamine, red staining) were observed between control (C) and NE-treated glands; in both cases, the dye diffused throughout the entire gland (Fig. 4C and E). These results are consistent with extensive gap junctional communication observed in pineal gland cells.

Figure 4: The entire pineal gland is highly coupled with GJ.

Figure 4:

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To evaluate the gap junctional communication among pineal cells in the whole pineal gland, we used a dye coupling technique in parallel with GFAP staining to examine the cell type that was coupled. A and B correspond to GFAP staining of glands treated with NE (10−4 M) and NE plus 18α-glycyrrhetinic acid (32 μM), respectively. Analysis of rhodamine diffusion in NE treated pineal glands for 15 min showed that the whole gland is coupled by GJ (C). The addition of 18α-glycyrrhetinic acid blocked the diffusion of sulforhodamine, and the staining captured primarily by the cells in the scrape area (D). Bar: 180 μm. (E) Corresponds to the quantification of the distance that the rhodamine diffused from the site of injury into another extreme of the gland. This diffusion was GJ dependent, as pre-incubation of the glands with octanol (O) or AGA prevented the dye diffusion. (F) Analysis of dye coupling in acute primary cultures of rat pineal gland cells indicates that most cells are highly GJ coupled after 6–8 h post culture. Application of NE did not affect the overall dye coupling. However, the application of O, AGA, Cx43p, or quinine reduced GJ communication to control levels. Scrambled peptide (Scr) did not alter GJ communication. * represent significant differences as compared to Control and NE treated conditions (p≤0.002, n=5–6 of experiments using different animal sets). # represent significant differences between O+NE or AGA+NE as compared to quinine (p≤0.001, n=4–6 of experiments using different animal sets, 120 animals were used for the analysis).

However, pretreatment of the glands with AGA restricted the dye diffusion to the small cut of the leptomeninges (Fig. 4D and E). No diffusion of dextran-FITC occurred; the compound remained at the site of the incision (data not shown). To evaluate the degree of gap junctional communication, dye coupling in the primary cells was performed. Confluent cultures were generated as described in experiments shown in Figure 2 (Fig. 4F). Cells (mostly pinealocytes and astrocytes) cultured for 6 to 8 h to allow for the formation of GJ communication reached almost 90% coupling (Fig. 4F, C for control conditions). Addition of NE for 15 min decrease GJ communication to 60% (Fig. 4F, NE). However, the application of GJ blockers, octanol or AGA, in control or NE conditions reduced GJ communication to 10% of the microinjected cells (Fig. 4F). Addition of the Cx43 blocking peptide (Cx43p) during the platting process reduced GJ communication to 20% (Fig. 4F). Addition of quinine to block Cx36 channels did not result in significant blockade of coupling compared to that obtained for NE condition (Fig. 4F) and did not alter the amount of NE-induced glycogen degradation (Fig. 2B). Scrambled peptide (Scr) did not affect dye coupling. Thus, GJ communication is high in confluent cultures, and GJ blockers are effective in reducing GJ communication.

The mechanisms of signaling amplification of NE-induced glycogen degradation mediated by GJ channels are calcium and cAMP-dependent.

To identify the mechanisms and second messengers involved in the NE-induced glycogen degradation, we examined the nature of the second messengers required for glycogen degradation and their GJ dependence using whole glands (Fig. 5A). No degradation of glycogen was observed in untreated conditions (Fig. 5A). NE treatment-induced glycogen degradation (Fig. 5A). Addition of β and/or α1 agonist, isoproterenol (ISO) and/or Phenylephrine (PHN) alone or in combination mimic the effects of NE in glycogen degradation (Fig. 5A). The preincubation of the glands with BAPTA-AM (5 μM) or Pertussis Toxin (10ng/ml, PT) and subsequent application of NE prevented the glycogen degradation induced by NE (Fig. 5A). Addition of calcium ionophore (A23187, 10 μM), 8Br-cAMP (1mM) or both also mimicked the effects of NE, indicating that a unique combination of β and α1 receptor activation for NE is required for efficient glycogen degradation (Fig. 5A). This results are in agrement with our previos publication that reduction of glycogen content induced by NE was dependent on the dual activation of β and α1 adrenoreceptors. The signal transduction of these receptors involves a cross talk between cAMP and Ca2+-dependent pathways to generate significant glycogen degradation in response to NE (Eugenin et al., 1997). Our data indicates that both, calcium and G coupled receptors can trigger glycogen degradation, however, the combination of both was more effective in inducing glycogen degradation in response to NE. In addition, blocking GJ communication before treatment with specific agonist ISO and PHN also prevented degradation of glycogen induced by NE (data not represented, 1.45±0.56 % reduction in glycogen content induced by ISO or PHN in the presence of gap junction blockers). Interestingly, the addition of the membrane permeable second messengers (A23187 and 8Br-cAMP) mimicked the results induced by NE and by the single and dual combination of ISO and PHN (Fig. 5A). Addition of BAPTA-AM or pertussis toxin to block transduction of α1 and β receptors respectively, prevented glycogen degradation induced by NE (Fig. 5A).

Figure 5: The mechanism of signaling and spread induced by NE and amplified by GJ is calcium and cAMP-dependent.

Figure 5:

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To determine the nature of the second messengers generated and transmitted via GJ, we quantified the glycogen degradation in response to different adrenergic and second messenger pathway agonists (A) in correlation with production of a key metabolic mediator of brain energy, lactate (B). (A) Determinations of glycogen content after 15 min of treatment with NE, Isoproterenol (ISO, 10−8 M), Phenylephrine (PHN, 10−8 M), A23187 (10 μM), 8Br-cAMP (1mM), or combinations of these agonists. All agonist-induced glycogen degradation in a GJ dependent manner because preincubation with O or AGA prevented glycogen degradation from controlling levels (1.45±0.56 %, n=7, in Reduction of Glycogen Content). Preincubation of the glands with BAPTA-AM, pertussis toxin (PT) or both prevented glycogen degradation induced by NE. (B) Determinations of lactate in the media of whole glands. As described in Figure A for glycogen degradation, most agonists resulted in lactate production, and combination of them resulted in additive effects. Blocking GJ communication with O or AGA prevented the secretion of lactate in response to NE, ISO, PHN, A23187, 8Br-cAMP, and the combination of these activators to control levels (1.089±0.05, n=5). * represent significant differences as compared to Control conditions (p≤0.005, n=6 of experiments using different animal sets, 168 animals were used for the analysis). # represent significant differences between agonist and blocker conditions as compared to NE conditions (p≤0.002, n=6 of experiments using different animal sets, 168 animals were used for the analysis).

Glycogen degradation induced by NE produces lactate in a GJ dependent manner.

To determine the metabolic product generated by the glycogen degradation induced by NE, we determined the amount of lactate generated after 15 min post-treatment in the medium of 5 whole pineal glands (Fig. 5B). NE induced significant production of lactate in the medium. Interesting activation of β or α1 adrenoreceptors with Isoproterenol (ISO) or Phenylephrine (PHN) did not result in comparable levels of lactate as compared to NE conditions (Fig 5B). However, the combination of both agonists resulted in same levels of extracellular lactate as compared to NE treatment (Fig. 5B). Blocking release of intracellular calcium or G protein activation using BAPTA-AM (5 μM) or pertussis toxin (10 ng/ml, PT), respectively, prevented the generation of lactate in response to NE treatment (Fig. 5B). Addition of a calcium ionophore, cAMP, or both recover the extracellular lactate levels to same levels observed with NE treatment (Fig. 5B). Blocking GJ communication with octanol or AGA prevented the secretion of lactate in response to NE, ISO, PHN, A23187, 8Br-cAMP and the combination of these activators to control levels (1.089±0.05, n=5–6, data not represented). Thus, GJ coordinates NE-generated glycogenolysis second messengers.

Discussion

The goal of this report is to document the complementation of the chemical and synapses to coordinate the entire glycogen metabolism of the pineal gland in response to adrenergic activation. Glycogen stores in astrocytes constitute an important energy source that rapidly metabolizes into the metabolic intermediaries required for high energetic metabolites, found in processes such as hormonal synthesis and secretion (Ishikawa & Shimazu 1980). In the pineal gland, astrocytes are the cells that store the major glycogen reservoirs (Eugenin et al. 1997). Interestingly, astrocytes are physically interposed between capillaries and neurons, suggesting that these cells could support neuronal metabolic activity (Bixel & Hamprecht 1995, Bouzier-Sore et al. 2002). The glycogen degradation in astrocytes results in glucose and lactate production that is essential to neuronal metabolism (see review (Medina et al. 1992a, Medina et al. 1992b)). Most enzymes involved in amino acid metabolism, carbohydrate metabolism, and taurine synthesis, glycogen phosphorylase to glycogen degradation, pyruvate carboxylase to the synthesis of oxalacetate and glycine cleavage system to glycine metabolism are expressed in astrocytes and not in neurons (see review; (Kirchhoff et al. 2001)). The heterogeneity of astrocytes and different neuronal populations supported in differential and sectored expression of enzymes involved in glucose or lactate degradation to obtain energy (Magistretti et al. 1993) support the idea that CNS areas are cooperatively coupled to maintain neuronal activity. Thus, communication and cooperation between both cell types are essential.

Moreover, the significant decrease in NE-induced glycogenolysis in dissociated pineal cells suggests that several cells may not respond to NE, possibly due to the lack of adrenergic receptors and the necessity of electrical communication. A similar phenomenon was reported in the vasopressin-induced glycogenolysis of rat hepatocytes where the glycogenolytic message is transmitted through GJs (Eugenin et al. 1998), from hepatocytes that express vasopressin receptors to hepatocytes that do not express the receptors but degrade glycogen (Eugenin et al. 1998). In hepatocytes, the transmitted signals may be constituted by a Ca2+ wave that propagates through GJs (Leybaert & Sanderson 2012, Gaspers & Thomas 2005, Niessen et al. 2000, Eugenin et al. 1998, Tordjmann et al. 1997). In pineal cells, NE may induce such Ca2+ wave through activation of α1 adrenoreceptor known to be coupled to phospholipase C and generate IP3, which may diffuse through GJs and induce the release of Ca2+ from calciosomes (Saez et al. 2003). A surprising unspecific effect was the negative regulation of eNE release induced by the electrical stimulus in the presence of octanol, probably by altering the activity of Ca2+ channels involved in the process of neurotransmitter exocytosis (Tachikawa et al. 1989). Octanol’s unspecific effect is not an issue in the case of exogenous application of NE because the release of the neurotransmitter is bypassed.

The robust increase in lactate detected in the extracellular medium correlated with the degradation of glycogen induced by NE and corroborate our published data that rat pineal gland had a unique cross-talk between β or α1 adrenoreceptors (Eugenin et al. 1997). The dependency of glycogen degradation and lactate production on calcium and cAMP is consistent between the agonists. Interestingly, lactate production was fully dependent on GJ communication. However, dye coupling was mostly dependent on Cx43 instead of Cx36, present on pinealocytes (the most abundant cell type in the pineal gland). In agreement, with the importance of Cx43 containing channels, but not Cx36 containing channels, for glycogen degradation (Fig. 2B), dye coupling (Fig. 4F) and signaling (Fig. 5) was fully dependent on Cx43 channels, but not Cx36. Our data indicate that most glycogen stores are in astrocytes (Eugenin et al., 1997), supporting the importance of Cx43 containing channels in spreading glycogenolytic signals across the entire pineal gland.

A similar mechanism of localized receptor expression and GJ amplification was described in the liver (Eugenin et al. 1998). Our publications indicated that the glycogenolytic response of the liver to vasopressin was mostly transmitted from the peri-central into the peri-portal acinus area. Several laboratories demonstrated that this metabolic cooperation was mostly mediated by Calcium and IP3 auto-regeneration, also termed calcium waves (Dupont et al. 2000a, Nathanson et al. 1999a, Tordjmann et al. 1997, Nathanson et al. 1995, Nathanson et al. 1994, Thomas et al. 1991, Rooney et al. 1990). Calcium waves can reach long distances and mostly present in cells expressing GJs. Indeed, GJs create an efficient syncytium enabling small second messengers such as calcium, IP3, and cyclic nucleotides to diffuse between connected non-excitable cells. In excitable cells (e.g., neurons), the major function of GJs is the fast relay of current generated at the plasma membrane by the exchange and intercellular diffusion of K+, Na+, and Cl− ions. We propose that in the pineal gland both systems are active and cooperative. In agreement, it has been described that Cx36 and Cx43 have particular and well-defined permeabilities that promote the spread of particular second messengers (Bukauskas 2012). We believe that these differences in GJ permeability could account for the dependency of glycogen degradation on Cx43 (only present in 10% of the cells, astrocytes), instead of Cx36 present in 90% of all cells, pinealocytes. Thus, the main framework of the metabolic changes in the pineal gland is driven by astrocytes communicated by Cx43.

The finding that small dyes that can only diffuse by GJ channels diffuse through astrocytes and pinealocytes in the whole pineal gland confirms that both cell types form extensive functional gap junctional networks, as it has been shown in isolated astrocytes and pinealocytes maintained in culture (Saez et al. 1991). Furthermore, the dye coupling found in the whole pineal gland was more extended than was found in cultured pineal cells (Saez et al. 1991, Saez et al. 1994), suggesting that GJs play a more relevant role than expected from results obtained in dissociated cells. In the pineal gland, pinealocytes and astrocytes may form independent networks, but under specific conditions they might interact, forming a unique network via heterotypic GJs. Our data support a physiological role for gap junctional communication in the CNS, which is to coordinate metabolic response and glycogen degradation in response to the exogenous and endogenous neurotransmitter, NE.

Acknowledgments.

This work was funded by The National Institute of Mental Health, grant MH096625, the National Institute of Neurological Disorders and Stroke, NS105584, and UTMB internal funding (to E.A.E). Also, the ICM-Economía P09-022-F Centro Interdisciplinario de Neurociencias de Valparaíso (to J.C.S.).

Abbreviations used:

AGA

18α-glycyrrhetinic acid

BAPTA

1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid

CaCl2

Calcium chloride

Ca2+

calcium ion

CNS

Central Nervous System

Cx

Connexin

DMSO

Dimethyl sulfoxide

EDTA

Ethylenediaminetetraacetic acid

eNE

endogenous noradrenalin

FBS

Fetal Bovine Serum

GABA

γ-aminobutyric acid

GFAP

Glial fibrillary acidic protein

GJ

Gap Junction

HPLC-EC

High Performance Liquide Chromatography with Electrochemical detection

IP3

Inositol triphosphate

KCl

Potassium chloride

NaCl

Sodium chloride

MgSO4

Magnesium sulfate

Na2EDTA

Ethylenediaminetetraacetic acid disodium salt

NaH2PO4

Monosodium phosphate

Na2S2O3

Sodium thiosulfate

NE

norepinephrine

O

octanol

OCT

Embedding medium for frozen tissue specimens to ensure optimal cutting temperature

PAS

periodic acid-Schiff staining

PBS

Phosphate Buffer Saline

PFA

Paraformaldehyde

RRID

Research Resource Identifier

Footnotes

Conflict of interest. The authors declare no competing interests.

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