Effects of rolipram and roflumilast, phosphodiesterase-4 inhibitors, on hypertension-induced defects in memory function in rats
Sobhana George Sugin Lal Jabaris a, Haridass Sumathy a, Ramadass Satiesh Kumar b, Shridhar Narayanan c, Sadagopan Thanikachalam a, Chidambaram Saravana Babu a,n
Abstract
Hypertension (HT) is a prevailing risk factor for cognitive impairment, the most common cause of vascular dementia; yet, no possible mechanism underlying the cognitive impairment induced by hypertension has been identified so far. Inhibition of PDE-4 has been shown to increase phosphorylation of cAMP-response element binding protein in the hippocampus and enhance the memory performance. Here, we examined the effects of PDE-4 inhibitors, rolipram and roflumilast, on the impairment of learning and memory observed in hypertensive rats. We used 2k-1c hypertensive model to induce learning and memory defects. In addition, mRNA expression of PDE-4 sub-types A-D was also assessed in the hippocampus tissue. Systolic blood pressure (SBP) was measured by tail-cuff method was significantly increased in 2k-1c rats when compared to sham operated rats; this effect was reversed by clonidine, whereas, PDE-4 inhibitors did not. PDE-4 inhibitors significantly reversed time induced memory deficit in novel object recognition task (NORT). Further, the retention latency on the second day in the elevated plus maze model was significantly shortened after repeated administration of rolipram and roflumilast. Plasma and brain concentrations of rolipram, roflumilast and roflumilast N-oxide were also measured after the NORT and showed linear increase in plasma and brain concentrations. The PDE4B and PDE4D gene expression was significantly enhanced in hypertensive rats compared with sham operated however PDE4A and PDE4C remained unaltered. Repeated treatment with PDE-4 inhibitors caused down regulation of PDE4B and PDE4D in hypertensive rats. These results suggest that inhibition of PDE-4 ameliorates HT-induced impairment of learning and memory functions.
Keywords:
Hypertension
Rolipram
Roflumilast
Roflumilast N-oxide
Cognition
1. Introduction
Hypertension is a prevailing vascular risk factor for cognitive decline and dementia, leading to a distressing loss of independence of the individual (Duron and Hanon, 2008). Several studies have focused on the possible mechanisms underlying the cognitive impairment induced by hypertension but a pathophysiological mechanistic link is still to be ascertained (Carnevale et al., 2012). The existence of memory deficits correlates with the presence of hypertension, and the subsequent pathological changes are generally foci of ischemic damage in deep cerebral white matter (Fazekas et al., 1993; Vermeer et al., 2003a). White matter damage may contribute to cognitive impairment and its most serious manifestation being dementia (Pantoni and Garcia, 1995; O’Brien et al., 2003; Vermeer et al., 2003b). People with controlled hypertension seem to have a lesser prevalence of white matter lesions than people with uncontrolled hypertension (Liao et al., 1996; de Leeuw et al., 2002).
Several clinical studies suggest that high blood pressure contributes to cognitive deficits in aging individuals (Elias et al., 1997; Skoog, 1997). Numerous pre-clinical models have shown positive (Wyss et al., 1992; Meneses et al., 1996; Meneses and Hong, 1998; Hirawa et al., 1999; Wyss et al., 2000; Hacioglu et al., 2003) but also negative (Kadish et al., 2001) correlations between hypertension and cognitive impairment in rats.
Phosphodiesterase-4 (PDE-4) is a critical regulator of intracellular level of cAMP that is expressed throughout the brain (PerezTorres et al., 2000). Activation of cyclic AMP (cAMP) signaling enhances memory function and synaptic plasticity (Wang et al., 2012). Therefore, targeting PDE-4 with a selective inhibitor may offer novel strategies in the treatment of memory impairment (Ghavami et al., 2006; Kodimuthali et al., 2008). A number of studies have identified that PDE-4 inhibition leads to elevation of cAMP levels (Barad et al., 1998), increased protein kinase A (PKA)/ phosphorylation of cAMP response element binding protein (pCREB) in the hippocampus signaling. This signaling cascade is essential for mediating memory, in particular hippocampus dependent long-term memory (Monti et al., 2006; Nagakura et al., 2002; Bourtchouladze et al., 2003). Rolipram, a specific PDE-4 inhibitor, has been shown to improve the working memory deficits caused by administration of scopolamine and MK-801in preclinical models (Zhang and O’Donnell, 2000; Zhang et al., 2000). Also, rolipram treatment reverses cognitive impairment and neuroinflammation induced by β-amyloid in rats (Wang et al., 2012). Very recently, brain penetrant selective PDE-4 inhibitor-GSK356278 demonstrated anxiolytic and improvement of cognition effects in preclinical models (Rutter et al., 2014).
Roflumilast is a second generation, highly selective PDE-4 inhibitor, and has recently been approved in several countries for severe COPD (Rabe, 2011). However, the role of PDE-4 inhibitors in improving learning and memory dysfunction induced by hypertension has not been studied so far. To evaluate the possibility of PDE-4 inhibitor as a therapeutic agent for cognitive dysfunction by hypertension, we examined the effects of rolipram and roflumilast on impairment of learning and memory function in the hypertensive rats using a twokidney one-clip (2k-1c) hypertensive model to induce deficits in learning and memory function.
2. Materials and methods
2.1. Animals
Adult male Wistar rats were obtained from Orchid Chemicals Pharmaceuticals Ltd, Chennai, India. Animals were housed in groups on soft bedding with food and water available ad libitum, in a temperature controlled environment with a light dark cycle of 12:12 h. All animals were allowed to habituate to the housing facilities for at least 1 week prior to surgery. Guidelines of “Guide for the Care and Use of Laboratory Animals” (Institute of Laboratory Animal Resources, National Academic Press 1996; NIH publication number #85-23, revised 1996) were strictly followed throughout the study. All experimental procedures were approved by the Institutional Animal Ethical Committee (IAEC), Sri Ramachandra University, constitute as per the directions of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India, (IAEC NO: IAEC/XXX111/SRU/258/2013).
2.2. Drugs
Rolipram and clonidine hydrochloride were purchased from Tokyo Chemical Industry Co., Ltd, Japan. Roflumilast was kindly gifted by Matrix Laboratories, Hyderabad, India.
2.3. Two-kidney one-clip induced hypertension in rats
Prior to surgical procedure, systolic blood pressure was measured by tail-cuff method (model MC 4000; Hatteras Instruments, Cary, NC, USA). Two-kidney one-clip induced Reno Vascular Hypertension (RVHT) was performed as per the method described previously with minor modifications (Zeng et al., 1998; Kalaivani et al., 2013). In brief, rats weighing 140–180 g at the time of surgery were anaesthetized by intra peritoneal injection of ketamine (75 mg/kg) and midazolam (1 mg/kg) mixture. A small incision was made and the left renal artery was exposed and cleared. Then a U-shaped silver clip with a gauge of 0.25 mm was placed around the renal artery and secured in place and the incision was sutured and the animals were returned to their cages.
In sham animals, incision was made, to expose the left renal artery and incision was sutured. Post-operative care was given to the rats for one week. The rats were maintained on drinking water containing 0.9% NaCl except sham group. SBP was measured starting three weeks after the renal artery constriction for 11 weeks. Animals were selected based on the SBP (cut off score Z170 mm Hg) at the 11th week post-surgery; selected animals were then stratified into groups based on their mean SBP, so that the mean baseline did not differ between the groups.
For testing the effects of repeated drug treatments on behavioral tasks such as novel object recognition task and transfer latency in elevated plus maze, vehicle (0.5% CMC) or clonidine (25 mg/kg., p.o), rolipram (0.03, 0.1, 0.3 mg/kg., i.p, dissolved in normal saline containing 5% DMSO), roflumilast (0.1, 0.3, 1 mg/kg., p.o) were given once per day for ten consecutive days. Here we selected clonidine, a α2 receptor agonist, centrally acting anti hypertensive drug to evaluate whether anti-hypertensive treatment would attenuate the cognitive impairments associated with HT in therapeutic model. For both behavioral experiments, separate set of animals were used including sham operated rats (68 for NORT and 66 for transfer latency; total no¼134). Both behavioral tests were performed 1 h following the treatment except rolipram group (45 min after dosing) as per the schedule (Fig. 1).
2.4. Novel object recognition task paradigm
The effects of repeated administration of PDE-4 inhibitors on recognition memory in hypertensive rats were assessed using NORT similar to that described previously (Prickaerts et al., 2002). Rats were acclimated to the open arena (40 60 60) without objects for 15 min. NORT consisted of two trial periods, acquisition trial (T1) and discrimination trial (T2) separated by a 24 h inter trial period. A rat was taken from its home cage and placed into the apparatus, central from the two objects, facing the wall in front of the observer, and the time spent actively exploring (exploration was defined as the animal sniffing, or touching it) the objects during a 3 min test period (T1) was recorded and returned to its home cage. The arena and test objects were cleaned with alcohol in order to remove any olfactory/taste cues. After 24 h, each rat was again placed in the test arena for recording T2 in the presence of one of the familiar object and a novel object, and the time spent exploring both objects were again recorded and discrimination index (DI) was calculated according to the following formula.
2.5. Transfer latency in elevated plus maze
The procedure and technique for testing learning and memory were followed as per the parameters described earlier (Reddy and Kulkarni, 1998; Hlinak and Krejci, 2002). The elevated plus maze for rats consisted of two open arms (50 cm 10 cm) and two closed arms (50 cm 10 cm 40 cm) extended from a central platform (10 cmof 50 cm from thefl10oor. On the 10th day of treatment, each rat wascm) and the maze was elevated to a height placed at the end of an open arm, facing away from the central platform. Transfer latency (TL) was defined as the time taken by the animal to move from the open arm into one of the closed arms with all its four legs was recorded. If the animal did not enter into one of the closed arm within 180 s, it was gently pushed into one of the two closed arms and TL was assigned as 180 s and rat was allowed to explore the maze for another 60 s. Then, the rat was taken out of the maze and returned to its home cage. The maze was cleaned thoroughly with 70% ethanol solution after each rat to remove any confounding olfactory cues. Retention latency (RL) was examined 24 h (11th day) after the transfer latency.
2.6. Pharmacokinetics of roflumilast, roflumilast N-oxide and rolipram after oral/i.p administration
Five min after the NORT experiment, rats were anaesthetized using isoflurane, and blood was collected retro-orbitally. After blood sampling, rats were euthanized by over dose of isoflurane and the brain was removed. Blood was centrifuged at 3000g for 10 min at 4 1C. Blood plasma and brain were frozen at 80 1C until compound level determination took place. The concentration of roflumilast and roflumilast N-oxide in plasma and brain were measured using a gradient LC/MS/MS procedure as described previously (Thappali et al., 2012). For rolipram, the brain samples were homogenized in 80:20 ratios of acetonitrile (ACN) and water with a tissue homogenizer. Two hundred micro liter plasma/brain homogenate were vortexed with 200 ml of ACN for 60 s. Then the samples were centrifuged at 10,000g for 15 min, 50 ml of supernatant was injected into the chromatographic system. The HPLC (Shimadzu Ltd., Japan) system consisted of a LC-20 AD binary pump, auto injector (SIL-20 AC HT), column oven (CTO-10 AS VP) and PDA detector (SPD-M20A). The wavelength of the detector was set at 230 nm. Detector output was quantified on Lab Solutions platform. Separation was carried out on a Enable C18G column 250 4.6 mm) using mixture of 0.5% formic acid and ACN (60:40) as a mobile phase, at a flow rate of 1 ml/min. Total analysis time was 15 min.
2.7. Corticosterone analysis
The serum concentration of corticosterone from the NORT experiment was quantified using enzyme-linked immunosorbent assay (Cayman Chemical, Ann Arbor, MI, USA).
2.8. Reverse transcriptase PCR
To determine the mRNA expression of PDE4 A, B, C and D in rat hippocampus was quantified using reverse transcriptase PCR (RT-PCR). Briefly, total RNA was extracted from hippocampus using TRIzol Reagent (Sigma). After homogenization, the tubes were incubated for 10 min and centrifuged at 100g for 5 min. 200 μl of chloroform was added to the supernatant, allowed to incubate for 5 min at room temperature and centrifuged at 12,000g for 20 min. Supernatant was transferred to new centrifuge tube to which 500 μl of isopropyl alcohol was added to precipitate the total RNA and centrifuged at 12,000g for 15 min following the incubation period of 10 min. The supernatant was decanted carefully and the pellet was washed three times with 75% ethanol and centrifuged at 12,000g for 15 min and the pellet was dried and resuspended in 20 μl of RNase free water. The concentration of RNA was determined using nanodrop spectrophotometer and RNA samples were stored at 80 1C until use. Two hundred nanogram of RNA were used for RT-PCR according to the manufacturer’s instructions (Genet Bio, Korea). Isolated RNA was allowed to undergo reverse transcription and polymerization reaction to synthesize cDNA using PCR master cycler gradient. The following steps were performed for each PCR reaction: 42 1C for 30 s, 94 1C for 5 min (1 cycle); 94 1C for 1 min, β-actin (55.4 1C), PDE 4A (71.0 1C), PDE 4B (59.7 1C), PDE 4C (58.1 1C) and PDE 4D (64.0 1C) and 72 1C for 1 min (with 35 cycles); and a final extension phase at 72 1C for 5 min. The concentration of cDNA was analyzed using nanodrop spectrophotometer. The RT-PCR products were loaded in 1% agarose gel and electrophoresed at 40 V for 30 min and bands obtained were quantified using gel documentation (Vilber, France). The primers are listed in Table 1.
2.9. Statistical analysis
Data are presented as mean7S.E.M. Statistical analysis was performed using GraphPad Prism Software (Version 5; Inc). The difference between time spent exploring the novel object versus familiar object during discrimination trial was calculated for each group and the level of significance was analyzed using two-sided student’s t test. For other parameters, the effect of sham vs vehicle was analyzed using a two-sided student’s t test and each treatment group vs vehicle group was analyzed using one-way analysis of variance (ANOVA) followed by Dunnet’s post hoc test. The level of significance was set at Po0.05.
3. Results
3.1. Effect of PDE-4 inhibitors on Blood pressure
The mean systolic blood pressure in male Wistar rats weighing 140–180 g which was approximately 120.879.8 mm Hg before renal artery occlusion, rose to 156.178.4 mm Hg at the end of three weeks, then exceeded 185.971.6 mm Hg at 11th weeks, respectively, after renal artery occlusion. To determine whether inhibition of the PDE-4 affects blood pressure in rats, SBP and responses to i.p/oral administration of rolipram and roflumilast, selective PDE4 inhibitors, were examined under the fully conscious state. As shown in Fig. 2(A), PDE-4 inhibitors did not cause a reduction in systolic blood pressure of the 2k-1c hypertensive rats, indicating that PDE-4 inhibitors had no therapeutic action on blood pressure. Clonidine, centrally acting α2 receptor agonist, at the dose of 25 μg/kg caused statistical significant reductions in SBP in rats on days 1, 5 and 9 were 149.773.1, 127.273.1 and 117.172.4 mm Hg, respectively. No significant change in SBP was observed during these experimental periods in vehicle treated rats.
Fig. 2(B)–(D) gives the mean kidney, heart weights, and heart index (heart weight normalized for body weight). The contralateral non-clipped kidneys had significantly higher weight when compared to clipped kidneys within the same group. As anticipated, hypertensive control showed significantly increased heart weights (1.4770.05 g vs 1.0370.01 g; Po0.001) and heart indexes (4.3070.14 vs 2.5070.12; Po0.001) compared with those in normotensive group. Both PDE-4 inhibitors caused no significant differences in heart weight and heart indexes compared with hypertensive control rats. Clonidine, however, caused a significant reduction of heart weight as well as heart indexes (Po0.05).
3.2. Novel object recognition task
The cognition-enhancing effects of repeated administration of PDE-4 inhibitors were shown by its aptitude to delay natural forgetting in rats after a 24 h interval in the NORT. Rats with repeated treatment of PDE-4 inhibitors before acquisition trial (T1) explored the novel object significantly more than the familiar one during discrimination trial (T2) respectively, showing an increase in memory retention. These effects of repeated administration of centrally acting anti- hypertensive drug (clonidine) were qualitatively similar to those obtained with the hypertensive control rats (Fig. 3(A) and (B)).
3.3. Transfer latency in elevated plus maze
Transfer latency of first day (on tenth day of drug treatment) reflected the acquisition of learning behavior of animal whereas RL of next day reflected improvement of both learning and memory. Vehicle and clonidine administered for 10 days orally did not have any significant effect when compared with sham operated group on RL of eleventh day in elevated plus maze. However, rolipram treated intraperitonally at 0.03, 0.1 and 0.3 mg/kg showed remarkable reduction (Po0.05 and Po0.01) in RL, indicating significant improvement in learning and memory. In addition, roflumilast at a dose of 0.1, 0.3 and 1 mg/kg., p.o also improved learning and memory in RL compared to vehicle treated group, which indicated an improvement in the elevated plus maze learning performance (Fig. 4(A) and (B)).
3.4. Rolipram, roflumilast and roflumilast N-oxide concentrations in rat plasma and brain
The effects of repeated treatment of roflumilast on the exposure rates of rolipram, roflumilast and roflumilast N-oxide (active metabolite of roflumilast) in plasma and brain samples are represented in Fig. 5(A)–(C). Rolipram treated animals showed dose dependent detectable levels of rolipram in plasma and brain, when the final dose was administrated 53 min before sampling. The rolipram concentrations were 87.2778.27, 248.18728.06, 584.55727.27 nM in plasma and 148.18720.10, 427.18713.32, 1016751.57 nM in brain at 0.03, 0.1 and 0.3 mg/kg., i.p respectively. Furthermore, low dose of roflumilast treated animals showed no detectable levels of roflumilast in plasma, whereas, detectable concentration of roflumilast N-oxide was found in plasma (14.4871.77 nM). In addition, mid and high doses of roflumilast showed linear concentrations in plasma (10.4870.89 and14.9472.43 nM) as well as brain (27.9371.88 and 125.96725.56 nM). The roflumilast N-oxide metabolite concentration was 86.9678.34 and 125.96725.56 nM in plasma and 25.5672.62 and 64.05713.63 nM in brain when the final dose was administrated 68 min before sampling, respectively.
3.5. Corticosterone concentration in rat
Fig. 6 shows serum corticosterone concentration in 2k-1c hypertension rats treated with vehicle, clonidine or PDE-4 inhibitors. Clipping significantly increased serum corticosterone levels in 2k-1c hypertension group when compared to sham operated group (3989729 vs 3420716 pg/ml; Po0.001). High doses of rolipram and roflumilast treatment promoted an additional increase in corticosterone levels. One-way ANOVA indicated that there was a significant effect of PDE-4 inhibition on serum concentration of corticosterone with both rolipram and roflumilast showing an increase (Po0.01 and Po0.05). However, clonidine produced no changes in serum corticosterone when compared with vehicle treated group.
3.6. Determination of PDE4ABCD mRNA expression
To verify the effects of PDE-4 inhibitors on hypertension- induced changes in PDE-4 subtypes in the hippocampus, we examined PDE4A, PDE4B, PDE4C and PDE4D mRNA expression in the hippocampus using the same rats, decapitated with isoflurane anesthesia after the behavioral test, by RT-PCR. In the case of PDE4B and D, chronic PDE-4 inhibitors treatment dose dependently decreased its hypertensive rats. In contrast, PDE4A and C mRNA expression levels were not changed between sham and hypertension as well as treatments (Fig. 7(A)–(E)).
4. Discussion
In the present study we evaluated the possibility of PDE-4 inhibition as a therapeutic agent for cognitive dysfunction by hypertension, we examined the effects of rolipram and roflumilast on impairment of learning and memory function in the hypertensive rats. Compared with sham operated group, systolic blood pressure, right kidney (unclipped), heart weight and the heart indices of 2k-1c induced hypertensive rats were found to be significantly increased; with significant elevation of serum corticosterone being observed. These results are in agreement with previous studies which show that serum level of corticosterone was higher in 2k-1c hypertensive compared with normotensive rats (Zamir et al., 1983), blood pressure, heart weight and right kidney weights were significantly higher in hypertensive group compared with sham rats (Hacioglu et al., 2003). The mechanism by which chronic hypertension is maintained in 2k-1c induced RVHT is poorly understood. Possible mechanism could be due to activation of renin-angiotensin system (RAS) and also an abnormal activity of the hypothalamic pituitary-adrenal system may play a role in the pathogenesis of 2k-1c model (Zamir et al., 1983; Morishita et al., 1991; Derkx and Schalekamp, 1994; Sadjadi et al., 2002; Weissheimer et al., 2012).
In the present study, we have demonstrated that 2k-1c induced hypertension produced deficits of learning and memory in novel object recognition task and transfer latency in elevated plus maze compared with sham operated rats. Transfer latency in elevated plus maze appear to be a spatial memory (Reddy and Kulkarni, 1998) and NORT to be a recognition memory model (Fernandez and Garner, 2007). The possible mechanisms by which hypertension could increase risk of cognitive impairment is still elusive. Perhaps, the possible risk factors causing cognitive impairment could be compromised vasoreactivity and consequent risk of brain hypoperfusion, blood–brain barrier dysfunction, amyloid plaques, increased risk of stroke, occlusion and degeneration of cerebral capillaries as well as the neuroinflammation (Blossom et al., 2008; Gentile et al., 2009; Peters, 2012; Carnevale et al., 2012).
In view of previous data demonstrating that second messengers such as cAMP play a prominent role in cellular signaling pathways and neuroplasticity, improve memory performance and based on the well established concept that cAMP elevations exert long-term potentiation (LTP) effects in hippocampus, we speculated that specific PDE-4 inhibitors may have beneficial effects in HT induced cognitive impairment by protecting against cerebral vascular dysfunction, cerebral hypoperfusion and neuroinflammation.
Here we demonstrate that the effects due to hypertension were reversed by repeated treatment with PDE-4 inhibitor- rolipram which was confirmed by both memory tests. These results are supported by findings from previous studies, in which rolipram reverses memory impairments in various type of animal models (Imanishi et al., 1997; Zhang and O’Donnell, 2000; Zhang et al., 2000; Nagakura et al., 2002; McLean et al., 2009; Li et al., 2011a, 2011b; Wang et al., 2012). In addition, a similar response has been observed in hypertensive rats with roflumilast treatment in which there was a failure of improvement in the memory functions induced by hypertension 60 min after oral administration of clonidine, which significantly decreased the SBP throughout the treatment periods at tested dose. Thus, clonidine showed slight activity on heart weight and heart index, suggesting that it has significant role in blood supply to the brain compared to PDE-4 inhibitors. However, more recently, Rutten et al., 2009 demonstrated that intraperitoneal injection of rolipram (0.03 mg/kg) improved the memory functions of rats without affecting cerebral blood flow and glucose utilization. To our knowledge, this is the first report to assess the effects of roflumilast, a selective PDE-4 inhibitor on memory enhancement. Several PDE-4 inhibitors have been identified; unfortunately, development of PDE-4 inhibitors for therapeutic purposes has been hindered by side effects such as emesis (Robichaud et al., 2001, 2002; Rennard et al., 2008; Rabe, 2011).
In our present study, we measured the concentrations of rolipram, roflumilast and roflumilast N-oxide, the active metabolite of roflumilast with similar potency and specificity to the parent compound (Bethke et al., 2006) in plasma and brain after the NORT experiment. Here we demonstrated the plasma and brain concentrations of rolipram, roflumilast and roflumilast –Noxide increased in dose dependent manner. Rolipram was detected in brain as well as plasma at all three doses while the concentration of roflumilast and its active metabolite levels were not at detectable in brain at low dose but treatment with low dose of roflumilast resulted in memory enhancement which was observed from the memory test. This could imply that improvements of cognitive effects of rolipram and roflumilast is probably explained by differences in potencies (Hatzelmann and Schudt, 2001; Hatzelmann et al., 2010), administration routes, roflumilast is rapidly converted by CYP3A4 and CYP1A2 to roflumilast N-oxide after oral administration (Bethke et al., 2007), resulting in different degrees of PDE-4 inhibition by these drugs.
It is well established that PDE-4 inhibitors such as denbufylline, rolipram and BRL 61063 results in an increased plasma corticosterone from the adrenal glands and also interacts with the hypothalamo-pituitary-adrenal (HPA) axis in rat at both the anterior pituitary and the hypothalamus (Hadley et al., 1996; Kumari et al., 1997; Kung et al., 2000). In the present study we found that repeated administration of high doses of PDE-4 inhibitors caused significant increase in serum corticosterone level in 2k-1c induced hypertensive rats, but had no effect on the corticosterone at lower doses. Studies on rodents indicate that stress or corticosterone administration can impair hippocampus specific learning and memory and also block hippocampal longterm potentiation (Kim et al., 2001; Alfarez et al., 2002; Woodson et al., 2003). In contrast, glucocorticoids have also been associated with enhanced memory (Sandi et al., 1997; Roozendaal, 2000; Buchanan and Lovallo, 2001). However, in our present study we observed that elevation of corticosterone due to PDE-4 inhibition failed to affect the behavioral tasks and that rolipram and roflumilast exerts a beneficial effects in HT induced cognitive impairment model is an important finding. Consistent with these findings, these PDE-4 inhibitors may be therapeutically useful for the enhancement of cognition associated with hypertension. Depending on the potency, specificity and the chronicity of use, PDE-4 inhibitors can be a nootropic.
In addition, we investigated the PDE-4 subtypes mRNA expression in hippocampus tissues. The PDE4 family consists of four genes in rats: PDE 4A, 4B, 4C and 4D (Houslay and Milligan, 1997), and its subtypes are distributed throughout the brain (PerezTorres et al., 2000; Bian et al., 2004) which indicate distinct roles of the individual PDE-4 in CNS functions. Previous study reported that PDE4A is highly expressed in rat hippocampus CA1 sub-region (Zhang et al., 2004). Whereas, PDE4B is the predominant present in the amygdala, hypothalamus, and striatum; it also expressed in frontal cortex and olfactory bulb (Zhang et al., 2008) as well as hippocampus (Dlaboga et al., 2006). While PDE4C is minimally expressed in the CNS (Zhang et al., 2004). In addition, PDE4D is also highly expressed in hippocampal CA1 region, mediating memory, its major role is cAMP hydrolysis, hippocampal LTP (Perez-Torres et al., 2000; Zhang et al., 2002; Rutten et al., 2008a) and antidepressant activity (Zhang et al., 2002). To date, few studies indicate that PDE4B may be involved in depression behavior, schizophrenia as well as anxiety (Millar et al., 2005; Zhang et al., 2008). On the finding that pharmacologically induced over expression of PDE4D resulted in memory impairment (Giorgi et al., 2004). In addition, mice deficient in PDE4D displayed memory enhancement in different type of behavioral tests and these effects were mimicked by repeated administration of rolipram in wild-type mice (Burgin et al., 2010; Li et al., 2011a, 2011b). Recently, GEBR-7b, a novel PDE4D selective inhibitor, demonstrated that increased hippocampal cAMP level, improved spatial, as well as object memory performance in the object recognition tests (Bruno et al., 2011). Here we have demonstrated subtype specific inhibitory properties of rolipram and roflumilast on PDE-4, in which PDE4B and PDE4D are significantly inhibited. PDE4A and PDE4C subtypes are not inhibited by rolipram, roflumilast and both are not affected by hypertension as well. Therefore, our results provide further evidence that PDE4B and PDE4D plays a critical role in the mediation of memory processes in brain regions such as the hippocampus. These results suggest that the antiamnesic effect of rolipram and roflumilast could be related to subtypes inhibition as well as elevation of cyclic AMP levels in hippocampus.
We showed that memory deficits induced by hypertension are likely correlated with increased PDE-4 activity in the hippocampus. These beneficial effects of rolipram and roflumilast appear to be directly attributed to its inhibition of PDE-4 in the brain, in particular the hippocampus, leading to subsequent increases in cAMP/PKA/CREB signaling (Impey et al., 1996; Rutten et al., 2008b). Further studies are needed to clarify this signaling cascade in hypertension associated cognitive impairments. Our findings provide an insight into the involvement of PDE-4 in hypertension associated neurochemical and pathological alterations of brain and the therapeutic profile of PDE-4 inhibitors. With respect to clonidine treatment, the present study did not allow a definitive conclusion, whether clonidine could resist against cognitive disorder associated with HT, because of the limited data as well as contradictory results of clonidine (Jäkälä et al., 1999).
In conclusion, the rolipram and roflumilast significantly attenuated memory deficits induced by hypertension. These observations may be attributed to an integrated network of cellular effects of the PDE-4 inhibitors in cognition. The precise role of the PDE-4 inhibitors on other cognitive models remains to be explored.
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