J Mol Neurosci (2008) 35:201–209 DOI 10.1007/s12031-008-9054-x
Expression of gLTP in Sympathetic Ganglia from Stress-hypertensive Rats: Molecular Evidence K. H. Alzoubi & A. M. Aleisa & K. A. Alkadhi
Received: 25 September 2007 / Accepted: 12 February 2008 / Published online: 3 April 2008 # Humana Press Inc. 2008
Abstract We previously reported behavioral and electrophysiological evidence indicating that superior cervical ganglia (SCG) from rats that developed hypertension as a result of chronic psychosocial stress expressed ganglionic long-term potentiation (gLTP) in vivo. In the present study, we present additional supportive evidence by measuring changes in protein levels of essential signaling molecules in ganglia from chronically stressed rats. We compared protein levels of essential, LTP-related signaling molecules in ganglia isolated from chronic stress-hypertensive rats, known to have expressed gLTP, with those of the same molecules in normal ganglia 1h after eliciting gLTP by high frequency stimulation (HFS) in vitro. Immunoblot analysis showed a significant increase in the levels of phosphorylated CaMKII, total CaMKII, nitric oxide synthase (NOS-1), and calmodulin in SCG from both chronically stressed rats and from normal rat ganglia in which gLTP was expressed by HFS in vitro. Additionally, there was a parallel reduction in calcineurin protein levels in ganglia from both groups. The present results confirm that ganglia from stressed rats have
K. H. Alzoubi Department of Clinical Pharmacy, College of Pharmacy, Jordan University of Science and Technology, Irbid, Jordan A. M. Aleisa College of Pharmacy, King Saud University, Riyadh, Saudi Arabia K. A. Alkadhi (*) Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, TX 77204-5515, USA e-mail:
[email protected]
expressed gLTP in vivo and that synaptic plasticity in sympathetic ganglia may involve a molecular cascade largely similar to that of LTP in the hippocampal CA1 region. Keywords Calcineurin . CaMKII . Immunoblotting . Heme oxygenase . gLTP . SCG . Nitric oxide synthase
Introduction Ganglionic long-term potentiation (gLTP) is a protracted enhancement of the nicotinic pathway that has been demonstrated both in vivo (Alonso-deFlorida et al. 1991; Bachoo et al. 1992; Bachoo and Polosa 1992) and in vitro (Brown and McAfee 1982; Briggs et al. 1985; Minota et al. 1991; Alkadhi et al. 1996; 2001a, b, 2005a, b; Alkadhi and Altememi 1997; Gerges et al. 2002). Expression of gLTP is independent of activation of cholinergic, adrenergic (Briggs et al. 1985), or adenosine (Hogan et al. 1998) receptors and does not involve changes in the acetylcholine (ACh) content of the ganglion (Briggs et al. 1985) or in the sensitivity of nicotinic ACh receptors (Briggs and McAfee 1988; Briggs et al. 1988). Uniquely different from other forms of synaptic LTP, both the induction and maintenance of gLTP require activation of 5-HT3 receptors (Alkadhi et al. 1996; see Alkadhi et al. 2005a for review) by serotonin, likely released upon high frequency stimulation (HFS) from serotonincontaining small intensely florescent (SIF) cells within the superior cervical ganglion (SCG; Hadjiconstantinou et al. 1982). Serotonin 5-HT3 receptors are present on neurons in the SCG of various species, including the rat (Hoyer et al. 1989; Morales and Wang 2002). As 5-HT3 receptor is highly permeable to calcium (Nichols and Mollard 1996), it is possible that activation of the 5-HT3 cationic channel–
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receptor complex in the ganglion creates a medium for a focused influx of calcium to increase its intracellular level to that required for activation of upstream enzymes including protein kinase C (PKC), calmodulin, and calcium– calmodulin kinase II (CaMKII) needed for expression of gLTP. In addition, many lines of evidence suggest the involvement of nitric oxide (NO; Sheng et al. 1993; Lin and Bennett 1994; Alkadhi and Altememi 1997; Altememi and Alkadhi 1999) and carbon monoxide (CO; Alkadhi et al. 2001a) in the expression of gLTP in mammalian sympathetic ganglia and the avian parasympathetic ciliary ganglion. Chronic psychosocial stress is associated with the onset and aggravation of ischemic heart disease and produces a greater rise in blood pressure in patients with labile hypertension than in normotensive subjects (Esler et al. 1977; Boone 1991; McEwen 1998). Stress is also related to sustained hypertension and increased risk of coronary heart disease due to enhanced activation of the sympathetic nervous system (Siegrist 2001). Both genetic and stressinduced hypertensions involve a significant neural component. Elevation of blood pressure in a psychosocial stress rat model can be reversibly prevented or normalized by treatment with 5-HT3 receptor antagonists to prevent expression of gLTP (Alkadhi et al. 2005b). Similar results were obtained in spontaneously hypertensive rats and obese Zucker rats where a stress component of the hypertension was blocked by 5-HT3 receptor antagonists (Alkadhi et al. 2001b; Gerges et al. 2002). We hypothesize that stress-induced sustained increases in central sympathetic outflow to sympathetic ganglia provides the repetitive activation of pre-ganglionic nerve required to induce gLTP. This would result in a sustained increase in sympathetic tone to the blood vessels, leading to hypertension (Alkadhi et al. 2005b; see Alkadhi and Alzoubi 2007 for review). In this work, we present molecular evidence in further support of the in vivo expression of gLTP in sympathetic ganglia isolated from chronically stressed rats. We measured levels of signaling molecules involved in the expression of gLTP in ganglia isolated from chronically stressed rats and compared those to levels of signaling molecules in ganglia isolated from normal rats 1h after induction of gLTP by HFS in vitro.
Materials and Methods Animals All animal experiments were carried out in accordance with the NIH Guides for Care and Use of Laboratory Animals and approved by the University of Houston’s Institutional Animal Care and Use Committee. Experiments were done in adult male Wistar rats (Harlan Inc., Indianapolis, IN).
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Induction of Psychosocial Stress Psychosocial stress was induced using an intruder model (Gerges et al. 2001; Gerges et al. 2003; Alkadhi et al. 2005b). Two groups of Wistar rats (250 g) were housed in two cages (five rats/cage). Each group was allowed to remain with the same cage mates for at least 1 week to allow animals to establish a social hierarchy within each group. The stress procedure was generated by daily random exchange of two animals from one cage to the other for a period of 4–6 weeks. This disrupts the social hierarchy; therefore, rats need to continually adapt to new stressful situations. This procedure produces stress indicated by a significant increase in blood pressure (Szilagyi 1991; Alkadhi et al. 2005b) and a 50% increase in blood corticosterone level (Gerges et al. 2001). Measurement of Blood Pressure Systolic blood pressure was measured by tail–cuff plethysmography, as explained previously (Alkadhi et al. 2001b, 2005b; Gerges et al. 2002), before removal of ganglia for immunoblot analysis. Electrophysiological Recording from Isolated Superior Cervical Ganglion SCG were rapidly dissected out and carefully desheathed in oxygenated (95% O2, 5% CO2) Locke solution (pH 7.4; NaCl 136 mM, KCl 5.6 mM, CaCl2 2.2 mM, MgCl2 1.2 mM, NaH2PO4 1.2 mM, NaHCO3 16 mM, glucose 11 mM) under a microscope. The ganglion was placed in a constant temperature (32.1 + 1°C) chamber (3 ml) and continuously superfused (1.3 ml/min) with Locke solution. The cervical sympathetic pre-ganglionic and the internal carotid postganglionic nerves were drawn into capillary stimulating and recording bipolar suction electrodes, respectively. Square wave supramaximal test stimuli (duration, 0.3 ms; Grass S88 stimulator) at 0.017 Hz were used to evoke compound action potentials (CAPs). Amplified CAPs were visualized on an oscilloscope, as well as on the monitor of a computerbased acquisition system (Digidata 1322A, Axon Instruments, CA), in conjunction with pCLAMP 8.2 software (Axon Instruments). After stabilization for about 30 min, baseline CAPs were recorded for 20 min, and thereafter, gLTP was evoked by HFS (20 Hz/20 s). Then, test stimuli were resumed, and CAPs were recorded and averaged every 5 min for at least 1 h. Changes in CAP amplitude were expressed as percentage of baseline CAP recorded before the train. Immunoblotting Animals in control and stressed groups were killed, and ganglia were immediately dissected out and homogenized in
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200 μl of buffered isotonic cocktail containing protease and phosphatase inhibitors (NaCl 150 mM, pepstatin 0.075 mM, leupeptin 0.1 mM, phenylmethylsulphonyl fluoride 1 mM, benzamidine 5 mM, ethylenediaminetetraacetic acid 1 mM, ethylene glycol tetraacetic acid 1 mM, Tris 20 mM, Na4P2O7 15 mM, B-glycerophosphate 100 mM, NaF 25 mM). Ganglia from the stimulated control group were subjected to the same procedure 1 h after expression of gLTP by HFS (20 Hz/20 s). The samples were then sonicated, and total protein was estimated by bicinchoninic acid assay (Pierce Chemical Rockford, IL). Samples were stored at −80°C until use. Just before use, samples were diluted [using the same buffer solution and sodium dodecyl sulfate (SDS) sample buffer] to contain the same concentration of protein (5 μg/20 μl) and then boiled for 5 min. Equal volumes of each sample were resolved in 8–16% SDS–acrylamide gel. Proteins on the gel were transferred to polyvinylidene fluoride membranes. Blots were first incubated with a primary antibody followed by the appropriate secondary antibody. For phosphorylated (P)-CaMKII, a mouse monoclonal antibody 22B1 (anti-P-CaMKII; 1:1,000 dilution; Affinity Bioreagent, Golden, CO), which recognizes CaMKII only when it is autophosphorylated at threonine 286, was used. The antigen–antibody complexes were visualized with HRP-conjugated goat anti-mouse IgG using electrochemiluminescence. Autoradiographs were generated, and bands were quantified by densitometry. Mouse polyclonal antibody to detect glyceraldehyde phosphodehydrogenase (GAPDH; 1:2,500 dilution; Sigma RBI) was used as a loading control. The ratio of test protein band intensity to GAPDH band intensity was compared among the different groups. For detection of total CaMKII, a rabbit polyclonal anti-CaMKII was used (1:1,500 dilution; Santa Cruz Biotechnology, Inc., CA). This antibody binds equally well to the phosphorylated and the non-phosphorylated forms of CaMKII; thus, total CaMKII levels could be estimated. PKCγ and PKCβ were probed using respective rabbit polyclonal antibodies for these molecules (1:500 dilution; Santa Cruz Biotechnology, Inc.). Calmodulin was detected using monoclonal mouse anti-calmodulin (1:500 dilution; Upstate Biotechnology, Lake Placid, NY). Calcineurin was detected using rabbit polyclonal anti-calcineurin antibody (1:1,000 dilution; Affinity Research Products). A rabbit polyclonal anti-NOS1 was used to detect nitric oxide synthase (1:1,000 dilution; Santa Cruz Biotechnology, Inc.). Heme oxygenase (HO2) was detected using a rabbit polyclonal anti-heme oxygenase 2 (1:2,500 dilution; Santa Cruz Biotechnology, Inc.). Statistical Analysis One-way analysis of variance was used to compare values among groups. Unpaired t test was used to compare two groups when needed. Significance of differences was set at
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p < 0.05. Values for immunoblots are represented as mean ± SEM; blood pressure values are expressed as mean ± SD.
Results Stress-Induced Expression of gLTP: Electrophysiological Evidence We have shown that due to occlusion, ganglia from stress hypertensive rats did not exhibit gLTP when subjected to HFS in vitro because the response has already been expressed in vivo as a result of chronic stress (Alkadhi et al. 2005b; see Alkadhi and Alzoubi 2007 for review). Previous reports from this lab showed that treatment of ganglia with 5-HT3 receptor antagonists caused inhibition of basal ganglionic transmission in ganglia from stressed rats without affecting basal transmission in those from control rats (Alkadhi et al. 2005b; see Alkadhi and Alzoubi 2007 for review). In this report, we repeated a series of experiments that measured the response to HFS of ganglia from stressed rats and compared it to that of ganglia from normal rats. The amplitude of the CAP at 1h after HFS was 131.3 ± 6.1% of baseline (n = 13) for ganglia from normal rats, but no significant change in the amplitude of CAPs was observed in stressed rats ganglia (102.4 ± 6.0%; n = 8, Fig. 1). Signaling Molecules Protein Levels Although the molecular mechanisms of LTP in the brain have been studied at length, there are no reports of direct measurement of signaling molecules involved in the induction and maintenance of gLTP in sympathetic ganglia. Synaptic plasticity in sympathetic ganglia may involve a molecular cascade similar to that of LTP of the brain hippocampal CA1 region. We measured, by immunoblotting, the protein levels of certain signaling molecules in SCG isolated from three groups: normal rats (control ganglia), chronically (4–6 weeks) stressed rats (stress ganglia), and normal rats where isolated ganglia were subjected to HFS to induce gLTP in vitro (S-control ganglia). In the latter group (nine ganglia), after in vitro HFS, the mean CAP amplitude, measured 60 min after HFS, was 132 ± 4.4% of the baseline ( p < 0.05), confirming expression of gLTP in stimulated ganglia from normal rats. Calmodulin and CaMKII According to our putative cascade of events leading to gLTP expression (Alkadhi et al. 2005a), presynaptic activity-induced elevated intracellular calcium binds to calmodulin, forming calcium/calmodulin (Ca/cam) complex, which activates CaMKII, forming P-CaMKII (Wang and Kelly 1995). Therefore, if gLTP had been already
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phosphorylation of kinases. Western blot analysis revealed a reduction of calcineurin protein levels in stimulated ganglia (69 ± 9%; Fig. 4). Similar decreases (56 ± 14%) were also seen in stressed ganglia, which strongly suggests expression of gLTP in these ganglia in vivo (Fig. 4). Protein Kinase C According to our putative molecular cascade of events leading to the expression of gLTP, HFSinduced calcium influx activates PKC, which, in turn, activates hemeoxygenase-2 (HO-2) to produce the CO necessary for the brief induction phase of gLTP (Alkadhi et al. 2005a). Immunoblot analysis revealed increases in the protein levels of PKCγ (157 ± 15%) and PKCβ (182 ± 20%) in stimulated ganglia compared to control ganglia (Fig. 5a,b). However, no change was observed in the levels of PKCg or PKCβ in stressed ganglia (Fig. 5a,b). Fig. 1 High-frequency stimulation of pre-ganglionic nerves (at time= 0) evoked robust gLTP in ganglia from a group of normal rats (n=13 from 8 rats; systolic blood pressure mean±SD=108±3.5 mmHg). The same protocol in another series of experiment on ganglia from a group of stressed-hypertensive rats (n=8 from 6 animals; systolic blood pressure mean±SD=147±8.3 mmHg) did not produce significant enhancement of transmission, suggesting occlusion due to expression of gLTP in vivo in these ganglia. Insets are CAPs from representative experiments recorded at 60 min after HFS; calibrations (0.5 mV/ 20 ms) apply to both traces. Points (mean±SEM) between the two asterisks are significantly (P
