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Rapid modulation of TRH and TRH-like peptide release in rat brain and peripheral tissues by prazosin
Peptides 32 (2011) 1666–1676
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Rapid modulation of TRH and TRH-like peptide release in rat brain and peripheral tissues by prazosin Albert Sattin a,b,c,e , Albert Eugene Pekary b,d,f,∗ , James Blood b a
Psychiatry Services, VA Greater Los Angeles Healthcare System, United States Research Services, VA Greater Los Angeles Healthcare System, United States c Departments of Psychiatry & Biobehavioral Sciences, University of California, Los Angeles, CA 90073, United States d Center for Ulcer Research and Education, VA Greater Los Angeles Healthcare System, United States e Brain Research Institute, University of California, Los Angeles, CA 90073, United States f Department of Medicine, University of California, Los Angeles, CA 90073, United States b
a r t i c l e
i n f o
Article history: Received 13 April 2011 Received in revised form 11 June 2011 Accepted 13 June 2011 Available online 28 June 2011 Key words: Post-traumatic stress disorder Thyrotropin releasing hormone Limbic system
a b s t r a c t Hyperresponsiveness to norepinephrine contributes to post-traumatic stress disorder (PTSD). Prazosin, a brain-active blocker of ␣1 -adrenoceptors, originally used for the treatment of hypertension, has been reported to alleviate trauma nightmares, sleep disturbance and improve global clinical status in war veterans with PTSD. Thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH2 ) may play a role in the pathophysiology and treatment of neuropsychiatric disorders such as major depression, and PTSD (an anxiety disorder). To investigate whether TRH or TRH-like peptides (pGlu-X-Pro-NH2 , where “X” can be any amino acid residue) participate in the therapeutic effects of prazosin, male rats were injected with prazosin and these peptides then measured in brain and endocrine tissues. Prazosin stimulated TRH and TRH-like peptide release in those tissues with high ␣1 -adrenoceptor levels suggesting that these peptides may play a role in the therapeutic effects of prazosin. Published by Elsevier Inc.
1. Introduction A noradrenergic basis for the symptomatology of post-traumatic stress disorder (PTSD) was suggested by the significant elevation of CSF norepinephrine (NE) and direct correlation of individual levels with scores on the Clinician-Administered PTSD scale for 11 combat veterans (p = 0.004; [22]). This concept was further supported by results of clinical trials with the adrenolytic drugs, prazosin and clonidine both of which were effective in reducing or arresting the principal symptoms of PTSD, implying pharmacological specificity via reduction of ␣1 agonism in the CNS [by prazosin: 7, 9, 53, 59]. There is a single post-mortem study of locus coeruleus (LC) neuron counts in three US veterans with likely war-related PTSD showing no overlap with the counts of NE neurons from 4 controls and a 47% reduction in mean count suggesting neurodegeneration and compensatory hypersecretion of NE [8]. PTSD is diagnostically distinct from Major Depression (MDD) yet there is significant symptomatic overlap as well as clinical co-morbidity [10]. As in PTSD, elevated NE has also been documented in CSF of melancholic Major Depressive
∗ Corresponding author at: VA Greater Los Angeles Healthcare System, Bldg. 114, Rm. 229, 11301 Wilshire Blvd., Los Angeles, CA 90073, United States. Tel.: +1 310 268 4430; fax: +1 310 441 1702. E-mail addresses: [email protected], [email protected] (A.E. Pekary). 0196-9781/$ – see front matter. Published by Elsevier Inc. doi:10.1016/j.peptides.2011.06.012
Disorder (MDD) [63]. Post-mortem NE neuron counts in LC were also reduced in depressive suicides (6 vs. 11 controls; [3]). But both the elevations of NE in CSF of patients and the reductions of NE neuron counts post-mortem appear to be more extreme in PTSD than in MDD from these data: 41% increase vs. 33% increase in CSF; 47% decrease vs. 23% decrease in LC. A noteworthy pathophysiological difference between these two diseases is the normality of circulating cortisol levels in PTSD vs. its sustained elevation in melancholic MDD but in CSF, cortisol was elevated in both PTSD and MDD [4,63]. A role for glutamate is implicated in PTSD, as well as for other disorders of anxiety and affect [24,36]. Preliminary evidence suggests that the anti-glutamatergic agents lamotrogine and memantine reduce PTSD symptoms [5]. Thyrotropin-releasing hormone (TRH) is an endogenous antidepressant, neuroprotective, anti-epileptic tripeptide that is one of many co-transmitters within glutamatergic neurons [26,31,33] which are involved in the pathophysiology of many neuropsychiatric disorders (reviewed by Sattin [54]). These and other functions of TRH should be distinguished from the traditional neuroendocrine role of hypothalamic TRH. Along with glutamate, TRH is releasable from HC ex vivo [30] and it has neuroprotective effects on glutamatoceptive neurons in vitro [29]. In contrast to the hypothalamus where TRH itself predominates, suprahypothalamic TRH (pGlu-His-NH2 ) is one among many TRHlike peptides with the general structure of pGlu-X-Pro-NH2 where
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Table 1 Control and post-prazosin injection serum values of corticosterone, free T4, total T3 and glucose. *(p < 0.05); **(p < 0.025); ***(p < 0.01) by one-way ANOVA vs. control (# higher than mean for Lewis rats by 2-tailed non-paired t test). Rat strain group
Corticosterone ng/ml
Total T3 ng/dl
Free T4 ng/dl
Glucose mg/dl
SD Control 2h 4h 6h
183 540 539 408
± ± ± ±
65# 36* 74* 175*
2.13 1.93 1.57 1.21
± ± ± ±
0.64 0.09 0.26 0.15
54 42 41 31
± ± ± ±
12 4 17 4*
116 381 246 146
± ± ± ±
7 90*** 70* 36
Lewis Control 2h 4h 6h
36 284 332 301
± ± ± ±
38 119* 32** 97*
2.29 1.61 1.57 1.27
± ± ± ±
0.14 0.30* 0.10** 0.18***
43 40 28 24
± ± ± ±
7 7 4* 1**
110 219 220 135
± ± ± ±
9 75* 40* 14
Table 2 Effects of prazosin (single ip injection) on HPLC peak areas corresponding to TRH and TRH-like peptide levels in various brain regions of male Sprague–Dawley rats involved in regulation of mood and behavior. Glu-TRH Nucleus accumbens 0.60 2h 1.47 4h 1.36 6h
Peak 2
TRH
Val-TRH
Tyr-TRH
Leu-TRH
Phe-TRH
Trp-TRH
0.62 1.19 ↑2.57**
0.52 2.47 3.46
0.13 1.35 0.97
0.46 1.97 1.57
1.00 3.09 2.18
0.12 2.61 0.08
0.45 4.15 4.03
1.18 0.46 1.32
2.29 1.76 1.98
0.92 0.76 ↑1.66**
↑3.16* ↑3.58* ↑2.55*
1.88 1.28 0.97
2.50 2.91 5.32
1.64 1.96 1.47
0.31 0.29 0.53
Piriform cortex 0.60 2h 4h 0.99 0.38 6h
0.70 0.50 ↓0.31
0.74 1.34 0.47
↑3.97* 1.55 0.52
2.12 0.96 0.42
1.82 0.70 0.50
↑3.05* 0.75 0.51
0.22 0.78 0.26
Frontal cortex 2h 0.64 4h 0.61 1.02 6h
2.08 5.53 2.43
1.86 2.19 2.19
2.44 ↑7.00** 1.85
0.96 ↑1.58* ↑1.89*
0.98 ↑2.89* 1.89
1.07 ↑4.25* ↑3.47*
1.24 0.80 ↑2.74*
Hippocampus 1.31 2h 0.64 4h 1.68 6h
1.42 1.51 2.34
1.01 1.42 2.82
↑2.18* ↑2.27* ↑2.16*
4.61 2.42 1.64
2.78 1.45 1.11
1.27 ↑4.75* 2.12
↓0.43* ↓0.26** ↓0.44*
Anterior cingulate ↑2.71* 2h ↓0.17* 4h 0.46 6h
0.62 ↓0.34* 0.89
1.60 0.20 2.30
1.85 0.12 0.37
↑3.76* 0.25 0.66
3.19 0.46 1.25
0.68 0.62 0.11
2.26 0.27 ↑4.76*
Posterior cingulate 0.88 2h 0.73 4h 0.60 6h
1.10 1.36 1.11
0.83 ↑3.13* 2.03
1.32 1.14 1.20
1.78 1.13 1.03
0.80 1.15 1.78
0.58 0.96 0.25
0.62 0.38 0.83
Entorhinal cortex 0.19 2h 2.07 4h 2.06 6h
0.50 1.08 ↑1.55*
0.47 1.02 ↑2.39*
0.65 2.02 ↑2.82*
1.63 ↑5.96* ↑5.37*
0.50 ↑3.97* 1.91
1.39 5.20 6.87
1.94 1.12 1.42
Striatum 2h 4h 6h
1.35 2.75 ↑3.82*
1.14 1.65 ↑4.08**
0.73 1.52 2.22
0.62 1.89 ↑2.49*
2.19 2.54 ↑4.84*
Amygdala 2h 4h 6h
0.85 0.86 1.78
0.80 1.50 ↑5.11***
0.61 1.40 1.17
Cerebellum 2h 0.87 ↓0.36** 4h ↓0.32** 6h
↓0.30*** ↓0.22*** ↓0.17**
1.82 2.22 1.78
0.96 0.70 0.84
0.49 0.87 0.46
4.08 1.36 1.53
0.87 0.81 0.79
0.87 0.46 0.56
Medulla oblongata 2h 0.38 1.13 4h – 6h
↓0.33* ↑1.98* 0.49
0.39 1.52 0.75
0.46 ↑3.28* 0.62
0.50 0.83 ↓0.21*
0.54 1.67 0.30
0.49 0.94 1.94
↓0.08* 1.30 ↓0.04*
Results are peak area divided by corresponding peak area in controls. *p < 0.05; **p < 0.025; ***p < 0.025 by one way ANOVA using post hoc Scheffe contrasts vs. the control group [47]. (–) Fractions lost during HPLC.
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Table 3 Effects of prazosin (single ip injection) on HPLC peak areas corresponding to TRH and TRH-like peptide levels in various brain regions of male Lewis rats involved in regulation of mood and behavior. Glu-TRH
TRH
Val-TRH
Tyr-TRH
0.66 0.90 1.39
1.12 0.95 ↑1.97*
1.37 ↓0.46* ↓0.34*
1.23 ↑3.00* 0.95
1.73 0.55 2.38
2.09 2.13 1.98
1.01 0.75 1.14
1.88 1.51 1.65
1.34 0.95 1.03
↑1.33* 0.82 ↑1.35*
1.37 2.13 2.74
0.59 0.57 0.75
0.79 1.34 0.74
1.66 0.73 1.19
1.40 0.66 1.34
Piriform cortex 0.64 2h 4h 0.54 6h 0.27
1.13 0.76 0.66
0.97 0.58 0.99
1.12 0.78 ↓0.24*
↑4.72* 0.89 0.22
1.88 1.15 1.21
1.00 0.52 0.45
0.68 0.46 0.60
Frontal cortex 2h 4h 6h
0.64 0.79 0.78
1.63 2.19 2.74
2.07 ↑5.63* 2.71
1.09 2.76 ↑7.04**
0.92 1.35 1.16
1.18 0.85 ↑2.23*
1.06 0.71 ↑3.57*
1.13 0.97 1.47
Hippocampus 2h 4h 6h
2.22 0.90 0.45
0.54 0.43 ↓0.25*
0.95 0.35 0.25
1.43 1.12 0.43
1.66 0.91 0.67
0.76 0.37 0.55
↓0.25* ↓0.22* ↓0.09**
↑2.98** 1.58 0.59
Anterior cingulate 1.15 2h 4h 1.12 ↑1.89* 6h
0.88 1.16 ↑3.06*
1.56 ↑6.03* 2.62
0.38 0.52 1.03
2.04 0.96 2.09
0.25 0.47 0.41
1.02 1.66 1.08
0.55 0.67 0.63
Posterior cingulate 1.26 2h 4h 0.87 6h 1.16
1.00 0.56 2.58
0.91 0.81 0.58
0.62 0.71 0.59
1.10 1.02 0.49
1.45 0.81 1.02
↑14.36** 1.15 0.78
2.04 1.07 1.22
Entorhinal cortex 0.61 2h 4h 1.04 1.30 6h
↓0.51* 0.61 1.04
1.01 ↑2.35* 2.24
1.54 2.43 ↑3.16*
0.91 1.12 ↑3.10*
1.12 1.75 1.69
1.22 1.65 3.73
0.93 0.94 1.75
1.17 0.84 1.03
1.14 0.65 0.97
1.04 0.81 1.14
0.91 0.52 1.74
1.02 0.84 2.14
1.19 0.71 0.87
1.33 1.13 1.36
0.64 ↓0.53* 0.84
1.25 ↓0.49* ↓0.47*
0.83 0.80 ↓0.49*
1.59 0.57 0.63
1.47 ↓0.34* 0.51
1.44 1.51 0.50
2.10 1.49 0.73
1.62 1.31 0.54
1.09 1.09 0.68
1.59 1.85 ↑2.90*
1.78 1.70 1.51
1.00 1.61 ↑2.79*
1.69 3.81 3.48
1.94 5.50 3.16
1.77 2.83 1.37
1.22 1.80 2.27
Nucleus accumbens 1.25 2h 1.09 4h 0.82 6h Amygdala 2h 4h 6h
Striatum 2h 4h 6h Cerebellum 2h 4h 6h
Medulla oblongata 1.60 2h 1.99 4h 1.41 6h
Peak 2
Leu-TRH
Phe-TRH
Trp-TRH
*p < 0.05; **p < 0.025; ***p < 0.025 by one way ANOVA using post hoc Scheffe contrasts vs. the control group [47].
X = any amino acid. This also occurs in many peripheral tissues. Phe-TRH (X = Phe) exhibits marked circadian rythmicity [47]. Neuroprotective effects of Glu-TRH were up to fourfold greater than TRH [29]. Both TRH and Glu-TRH are augmented in brain after electroconvulsive seizures [54]; TRH confirmed by Newton et al. [37]. Psychopharmacological agents and the photoperiod [41,46,47,50], thyroid hormones, glucocorticoids, GSK-3, lipopolysaccharide, leptin, and ghrelin also modulate these tripeptides in brain and peripheral tissues. Leptin and ghrelin evoke the most rapid alterations of these peptides in brain and periphery suggesting their involvement in downstream effects, including their antidepressant effects [43,48]. In the present study we compare the effect of prazosin on the expression of TRH and TRH-like peptides (pGlu-X-Pro-NH2 ) in the brain and peripheral tissues of young adult male Lewis and SD rats, two strains that have been reported to have high and moderate Extreme Behavioral Responses (EBR) to predator scent, respectively, an animal model of PTSD [11,12].
2. Materials and methods 2.1. Animals Male Sprague–Dawley (SD) and Lewis rats (Harlan, Indianapolis, IN) were used for all experiments. These animals were group housed (4 animals per cage), maintained with standard Purina rodent chow #5001 and water ad libitum during a standard one week initial quarantine in a controlled temperature and humidity environment; lights on: 6 a.m. to 6 p.m. All animals were weighed on the day of receipt and on the morning of each experiment. Initial and final body weights did not differ between experimental groups. Research was approved by the VA Greater Los Angeles Healthcare System Animal Care and Use Committee and conducted in compliance with the Animal Welfare Act and the federal statutes and regulations related to animals and experiments involving animals and adhere to principles stated in the Guide for the Care and use of Laboratory Animals, NRC Publication, 1996 edition. All efforts
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Table 4 Concentration-dependent effects (0, 0.01, 0.1 and 1.0 × 3.9 mg/kg) 4 h after a single ip injection of prazosin on TRH and TRH-like peptide levels in various brain regions involved in mood regulation of male SD rats. Glu-TRH Nucleus accumbens 0.51 0.01 0.86 0.1 0.66 1.0
Peak 2
TRH
Val-TRH
Tyr-TRH
Leu-TRH
Phe-TRH
Trp-TRH
0.87 ↑2.27** 0.69
1.22 1.73 1.84
1.40 1.47 0.84
0.91 ↑2.35* 0.56
0.27 0.96 1.18
0.40 ↑3.41** 1.19
1.06 1.05 2.02
Amygdala 0.01 0.1 1.0
0.71 0.85 0.77
0.47 0.74 0.56
1.30 0.99 0.99
0.45 0.58 0.54
0.48 0.62 0.44
0.52 0.79 0.76
0.62 1.00 1.21
0.92 1.08 0.54
Piriform cortex 0.01 0.1 1.0
0.74 0.45 0.36
1.81 0.55 0.64
1.63 0.98 0.84
1.19 ↓0.31* 0.52
1.57 0.65 0.56
1.66 0.50 0.73
1.52 0.79 0.54
1.34 0.57 0.72
Frontal cortex 1.25 0.01 1.20 0.1 ↓0.32* 1.0
1.73 1.77 0.76
2.15 0.59 0.40
1.62 0.40 0.24
↑1.91** ↓0.49* ↓0.51*
↑1.94* 0.43 0.32
1.39 0.59 0.42
1.31 0.44 ↓0.27*
Hippocampus 0.01 0.1 1.0
0.94 0.45 0.48
2.42 1.07 1.8
1.45 0.66 0.68
1.80 0.51 0.70
0.98 0.40 0.39
0.55 ↓0.16* 0.22
0.96 ↓0.24* 0.38
Anterior cingulate ↓0.38* 0.01 0.1 0.93 ↓0.03* 1.0
1.26 0.57 0.49
1.02 0.50 0.32
0.79 ↓0.20* ↓0.15*
0.96 ↓0.32* ↓0.19*
0.53 0.30 0.20
1.35 0.38 0.28
1.41 0.73 0.64
Posterior cingulate 0.98 0.01 0.1 0.27 1.0 0.92
0.78 1.16 2.10
0.62 1.17 1.30
0.54 0.93 1.30
0.38 1.53 1.45
0.48 0.72 1.03
1.37 1.53 1.98
0.84 1.55 1.58
Entorhinal cortex 0.76 0.01 0.1 0.80 0.96 1.0
↓0.61* ↓0.41* 0.85
1.28 0.51 1.40
1.41 0.60 0.96
1.54 0.58 1.07
0.85 0.50 0.89
1.00 1.10 1.02
1.49 1.10 2.21
0.81 0.59 0.77
0.82 0.55 0.43
↓0.54* ↓0.18** ↓0.34**
1.16 0.50 0.48
↓0.29* ↓0.24* ↓0.26*
↓0.56* ↓0.30** ↓0.23**
↑2.98** 1.70 ↑1.96*
↑1.97* 1.36 ↑2.11*
↑3.89* 1.74 ↑3.35*
1.39 0.63 1.28
2.46 2.11 2.25
1.79 1.22 2.21
↑3.44* 1.73 ↑3.60*
2.26 1.61 2.19
↓0.33** 0.76 ↓0.00***
↓0.30* 0.37 ↓0.36*
↓0.27* 0.55 ↓0.49*
0.61 0.87 0.51
↓0.18* 0.41 0.27
↓0.05* ↓0.27* ↓0.09*
↓0.13* ↓0.29* ↓0.19*
Striatum 0.01 0.1 1.0 Cerebellum 0.01 0.1 1.0
0.64 0.47 0.43
Medulla oblongata 0.68 0.01 0.87 0.1 0.66 1.0
↓0.64** ↓0.14*** ↓0.08***
0.46 ↓0.25** ↓0.16**
*p < 0.05; **p < 0.025 by one way ANOVA using post hoc Scheffe contrasts vs. the control group as previously described [47].
have been made to minimize the number of animals used and their suffering. All animals were transferred from the Veterinary Medical Unit to the laboratory 12 h before the start of experiments to minimize the stress of a novel environment.
such that decapitation could occur at 20 min intervals between 9 a.m. and 11 a.m. to minimize the effect of the circadian rhythm on TRH and TRH-like peptide levels in rat brain [47]. 2.3. Pharmacokinetic properties of prazosin in rat
2.2. Ip vs. oral administration of prazosin Prazosin was administered ip rather than orally because firstpass metabolism of prazosin is partially responsible for its low oral bioavailability. Hepatic metabolism of prazosin accounts for more than 95% of its elimination, almost all of the metabolites are excreted in bile without re-entry into the systemic circulation [28]. The plasma clearance times for prazosin following oral and ip administration in normal human subjects are identical, 2.9 ± 0.56 and 2.9 ± 0.75 h, respectively [28]. Animals were injected at times
The relative amount of the injected dose of prazosin taken up by tissues is liver > kidneys > plasma = lungs > heart > spleen > brain. The percentage in brain is 4%. The clearance time (t1/2 ) of 44 min in rats does not differ significantly among tissues or plasma [16]. 2.4. Time-dependent effects of prazosin on TRH and TRH-like peptide levels in brain of SD rats Male SD rats, weighing 346 ± 17 g, were either uninjected (control group), or received a single 1.0 ml ip injection of 1.3 mg
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Table 5 Concentration-dependent effects (0, 0.01, 0.1 and 1.0 × 3.9 mg/kg) 4 h after a single ip injection of prazosin on TRH and TRH-like peptide levels in adrenal, pancreatic and reproductive tissues of male SD rats. Glu-TRH
Peak 2
Adrenal 0.01 0.1 1.0
TRH
Val-TRH
Tyr-TRH
Leu-TRH
Phe-TRH
Trp-TRH
↓0.24* ↓0.12** ↓0.30*
↓0.13** ↓0.47* ↓0.43*
0.63 0.73 0.52
0.63 0.66 0.66
1.04 1.76 0.53
0.9 1.42 0.67
0.91 1.40 0.47
1.33 ↑2.27* ↓0.41*
Pancreas 0.01 0.1 1.0
↓0.24** 0.78 ↓0.11**
0.51 ↓0.35* 0.68
0.52 1.10 1.02
0.81 0.78 0.8
↓0.41* 0.66 0.74
0.53 1.37 0.83
0.53 0.88 ↓0.28*
0.52 1.17 0.80
Testis 0.01 0.1 1.0
1.95 ↑2.62* ↑9.09***
0.41* 1.63 ↑2.49*
0.56 1.76 1.98
0.50 1.84 ↑4.12**
↓0.42* 1.43 1.60
↓0.36* 1.20 1.50
↓0.19** 1.14 0.98
↓0.29* 1.92 1.71
Epididymis 0.01 0.1 1.0
0.55 ↓0.40* ↓0.42*
↓0.19** ↓0.14** ↓0.21**
0.74 ↓0.27* ↓0.34*
0.50 ↓0.29* ↓0.36*
0.58 ↓0.30* ↓0.37*
↓0.34* ↓0.15** ↓0.29*
↓0.34* ↓0.15** ↓0.22**
↓0.31* ↓0.31* ↓0.23**
Prostate 0.01 0.1 1.0
↓0.32* ↓0.06*** ↓0.03***
↓0.41* ↓0.32* ↓0.34*
↓0.30* ↓0.49* ↓0.48*
↓0.38* ↓.17** ↓0.13**
↓0.37* ↓0.39* ↓0.24**
↓0.19** ↓0.17** ↓0.16**
↓0.25* ↓0.16** ↓0.12***
↓0.21** ↓0.36* ↓0.23**
*p < 0.05; **p < 0.025 by one way ANOVA using post hoc Scheffe contrasts vs. the control group as previously described [47].
Fig. 1. Concentration-dependent effects 4 h after a single ip injection of prazosin on TRH and TRH-like peptide levels in striatum of male SD rats. Note the progressive fall in TRH, Tyr-TRH, Leu-TRH, Phe-TRH and Trp-TRH levels with increasing prazosin concentration.
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Fig. 2. Concentration-dependent effects 4 h after a single ip injection of prazosin on TRH and TRH-like peptide levels in medulla oblongata of male SD rats. TRH and Peak 2, Val-TRH, Leu-TRH, Phe-TRH and Trp-TRH levels were significantly suppressed by 0.01× the maximum prazosin dose.
prazosin (3.8 mg/kg body weight) (Sigma, St. Louis, MO)/ml of 50% ethanol + 50% dimethyl sulfoxide 2, 4 or 6 h (n = 4 for each time point) before decapitation.
2.5. Time-dependent effects of prazosin on TRH and TRH-like peptide levels in brain of Lewis rats Male Lewis rats, weighing 258 ± 13 g, were either uninjected (control group), or received a single 0.7 ml ip injection of 1.5 mg prazosin (4.1 mg/kg body weight) (Sigma, St. Louis, MO)/ml of 50% ethanol + 50% dimethyl sulfoxide 2, 4 or 6 h (n = 4 for each time point) before decapitation.
2.7. Dissection of rat brain, pancreas, adrenals and reproductive organs After decapitation, nucleus accumbens (NA), amygdala (AY), frontal cortex (FCX), cerebellum (CBL), medulla oblongata (MED), anterior cingulate (ACNG), posterior cingulate (PCNG), striatum (STR), piriform cortex (PIR), hippocampus (HC), entorhinal cortex (ENT), pancreas, prostate, epididymis, testes and adrenals were hand dissected, weighed rapidly, and then extracted as previously described [41,44–51,55]. In a separate experiment, 4 rats were decapitated, the adrenals removed, and each adrenal cortex and medulla separated, with the aid of a dissecting microscope, and extracted as described above.
2.6. Concentration-dependent effects of prazosin on TRH and TRH-like peptide levels in brain and peripheral tissues of SD rats
2.8. Serum hormone assays
Male SD rats, weighing 257 ± 64 g, were injected with 0.5 ml of 50% DMSO + 50% ethanol containing 0, 0.01, 0.1 or 1.0 × 2.0 mg prazosin/ml (mean of 3.9 mg/kg prazosin at the highest dose) followed 4 h later by decapitation. The lowest dose corresponds to the typical 2–5 mg oral dose in patients.
Serum glucose was measured with the OneTouch Ultra Blood Glucose Monitoring System, Life Scan, Milpitas, CA. Serum corticosterone (CORT), free T4 , and total T3 were measured with the following commercial RIA kits: CORT (MP Biomedical, Aurora, OH), free T4 and total T3 (DPC Coat-A-Count, Los Angeles, CA). Rat insulin
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Fig. 3. Concentration-dependent effects 4 h after a single ip injection of prazosin on TRH and TRH-like peptide levels in epididymis of male SD rats. TRH and all TRH-like peptide levels fell progressively with increasing prazosin concentration.
and rat leptin RIA kits were purchased from Millipore, St. Charles, MO.
3. Results 3.1. Serum values of corticosterone, free T4 , total T3 , and glucose for time-dependent effects in SD rats
2.9. HPLC and RIA procedures, HPLC peak identification and quantitation HPLC and RIA procedures, peak identification, and quantitation by co-chromatography with synthetic TRH and TRH-like peptides, relative potency analysis of multiple antibodies to TRH and TRHlike peptides, mass spectrometry and resolution of overlapping peaks by least squares fitting of a 2-Gaussian statistical model have been previously reported in detail [41,44–51,55]. “Peak 2” is a mixture of at least 2 unidentified TRH-like peptides.
2.10. Statistical analysis Statistical comparisons were made with the aid of Statview (Abacus Concepts, Inc., Berkeley, CA), a statistical software package for the Macintosh computer. All multi-group comparisons were carried out by one way analysis of variance using post hoc Scheffe contrasts with the control group [44,47,48].
Serum corticosterone levels in SD rats increased 3-fold at 2 h followed by a progressive decline to a 2.2-fold increase at 6 h post injection. Total T3 levels decline progressively to 43% of the control value by 6 h due, in part, to the blockade of sympathetic nervous system stimulation of the type 2 deiodinase in brain and brown fat which converts T4 to T3 [52,57]. Glucose rose 3.3-fold at 2 h followed by a progressive decline to control values at 6 h. This rise can be attributed to a decrease in glucose uptake in muscle [27] following ␣1 -adrenoceptor blockade by prazosin and stimulation of corticosterone release [49] (see Table 1 and Section 3.8). 3.2. Serum values of corticosterone, free T4 , total T3 , and glucose for time-dependent effects in Lewis rats Serum corticosterone levels in Lewis rats were increased: 8(2 h), 9.2- (4 h) and 8.4-fold (6 h). Serum free T4 declined 30% (2 h), 31% (4 h) and 45% (6 h) while serum total T3 fell 35% (4 h) and 44% (6 h) due to increased glucocorticoid inhibition of pituitary TSH
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Fig. 4. Concentration-dependent effects 4 h after a single ip injection of prazosin on TRH and TRH-like peptide levels in prostate of male SD rats. TRH and all TRH-like peptide levels fell progressively with increasing prazosin concentration.
release decreased sensitivity to HY TRH release [61], and decreased noradrenergic stimulated conversion of T4 to T3 [57]. However, the absolute increase in corticosterone and glucose in Lewis rats was less than for the SD rats, despite a slightly higher dose of prazosin (see Table 1 and Section 3.8).
3.3. Overview of HPLC results for time-dependent effects in SD and Lewis rats The acute effect of a single ip injection of prazosin on levels of TRH and TRH-like peptides in brain and peripheral tissues was, in general, heterogeneous. Depending on the particular peptide and tissue or brain region, responses in peptide levels ranged from (a) no effect, (b) a rapid and sustained rise, (c) a rapid rise followed by a gradual decline toward the control value, (d) a rapid and sustained decline, or a rapid decline followed by a recovery to the control value. A significant decrease in peptide level at 2 h is consistent with acceleration of release [38]. Rapid change in TRH and TRH-like peptide levels was not a nonspecific response to the acute stress of the ip injections administered at 2, 4 and 6 h before decapitation. For example, ip injection of 14.1 mg/kg corticosterone resulted in a serum level of 27,806 ng/ml corticosterone at 2 h. Nevertheless, there were no
significant changes in TRH or TRH-like peptide levels in brain or peripheral tissues at 2 h compared to uninjected controls [49]. 3.4. Time-dependent effects of prazosin at 2 h in brain of SD rats Peak 2 and Trp-TRH levels in MED decreased 67% and 92%, respectively, by 2 h consistent with prazosin-induced release of these peptides (see Table 2). We abbreviate this result as: MED (2↓). Significant changes in TRH and TRH-like peptide levels for other brain regions were: ACNG (2↑), PIR (2↑), HC (1↓, 1↑), CBL (1↓), AY (1↑), for a total of 10 (Table 2). 3.5. Time-dependent effects of prazosin at 4 and 6 h in brain of SD rats Significant changes were: FCX (7↑), ENT (6↑), STR (5↑), HC (2↓, 3↑), MED (2↓, 2↑), AY (3↑), PIR (1↓), NA (1↑), PCNG (1↑), for a total of 33 (Table 2). 3.6. Time-dependent effect of prazosin at 2 h in brain of Lewis rats HC (1↓, 1↑), AY (1↑), PIR (1↑), PCNG (1↑) and ENT (1↓) changes totaled 6 (Table 3).
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Fig. 5. Comparison of the TRH and TRH-like peptide levels in the adrenal cortex and medulla of male SD rats. Note, for example, that TRH is 65% higher and Trp-TRH is 57% lower in the cortex compared to the medulla.
3.7. Time-dependent effect of prazosin at 4 and 6 h in Lewis rat brain
3.10. Concentration-dependent effects of prazosin in reproductive and pancreatic tissues
CBL (4↓), FCX (4↑), NA (2↓, 2↑), HC (3↓), ACNG (3↑), ENT (3↑), MED (2↑), AY (1↑), PIR (1↓), STR (1↓), totaled 26 (Table 3).
Prazosin at 0.01, 0.1 and 1.0 times the maximum dose had a profound suppressive effect on the levels of TRH and TRH-like peptide levels (increased release) in prostate and epididymis of SD rats 4 h after ip injection of prazosin (Table 5; Figs. 3 and 4).
3.8. Serum values for glucose, insulin, leptin, free T3, free T4, corticosterone, testosterone, for concentration-dependent effects
4. Discussion Serum leptin, free T3 , free T4 , corticosterone and testosterone levels were not significantly changed by increasing dose of prazosin vs. the vehicle (control) 4 h after injection (results not shown). The corresponding serum glucose values (mean ± SD, mg/dl) were: 65 ± 8 (control); 146 ± 40 (0.01× dose, n.s.); 132 ± 20 (0.1× dose, n.s.); 255 ± 130, (1× dose, p < 0.05). Serum insulin (mean ± SD, ng/ml): 0.05 ± 0.00 (control); 0.05 ± 0.00 (0.01× dose, n.s.); 0.08 ± 0.05 (0.1× dose, n.s.); 0.63 ± 0.64, (1× dose, p < 0.05). 3.9. Overview of concentration-dependent HPLC results in SD brain after ip prazosin Because of the limited solubility of prazosin in water and ethanol, DMSO is the solvent most commonly used for its in vivo administration. DMSO, however, has been reported to have a highly inhibitory effect on acetylcholinesterase at all concentrations tested [39]. As a result, the time-dependent effects of a single ip injection of prazosin in DMSO should be a combination of the prazosin and DMSO effects. Rather than performing a separate, time-dependent, control experiment involving the injection of DMSO alone, a concentration-dependent study of the prazosin effects at 4 h was carried out. This time point provided more significant responses than 2 h. Measurement of the TRH and TRH-like peptide responses to 0, 1, 10 and 100% of the time-dependent dose for prazosin should identify those tissues most sensitive to the effects of prazosin and properly control for any DMSO effects. Even though all of the concentration-dependent effects were obtained at 4 h after ip injection of prazosin it is possible to attribute changes in peptide levels at this time to altered release/degradation since ␣1 -adrenoreceptors regulate TRH secretion but not proTRH biosynthesis and processing [38] (see Tables 4 and 5; Figs. 1 and 2). The significant changes in TRH and TRH-like peptide levels for all prazosin concentrations tested were: STR (14↓); MED (13↓); CBL (8↑); ACNG (6↓); FCX (5↓,1↑); NA (3↑); HC (2↓); ENT (2↓); PIR (1↓); PCNG (0).
The most striking observation in the present study was the nearly quantitative release/clearance of all TRH and TRH-like peptides from the prostate and epididymis at even a very low dose of prazosin 4 h after injection. Contraction [60] and growth [34] of the prostate and epididymis are activated by noradrenergic sympathetic innervation of the membrane-bound and intracellular ␣1 -adrenoceptors found throughout the ejaculatory system of rats and man [13]. Prazosin and other ␣1 -adrenoceptor inhibitors are used for the treatment of the urologic complications of benign prostatic hypertrophy [13]. These particular adrenoreceptors are also responsible for stimulating the release of TRH from the hypothalamus in response to cold, and inhibiting its release in response to stress, but they do not modulate its synthesis, as is the case for the -adrenoceptors [38]. The norepinephrine concentration in the rat reproductive system, which is comparable to that of cortex and other extrahypothalamic brain regions [40] appears to provide an inhibitory tone for TRH and TRH-like peptide release which is reversed by ␣1 -adrenoceptor inhibitors such as prazosin. The total TRH immunoreactivity of the rat prostate is comparable to that of the rat hypothalamus [42]. The TRH-like peptides Glu-TRH and Leu-TRH were elevated 25- and 22-fold, respectively, in the epididymis 2 h after injection of escitalopram, a highly selective serotonin reuptake inhibitor [55]. Glu-TRH is a fertilization promoting peptide [20]. The present results and previous studies [35,50] suggest that TRH and TRH-like peptides participate in the regulation of reproductive functions by neurotransmitters such as norepinephrine and serotonin. Prazosin blocks the ACTH and CORT responses to the stimulation of the central nucleus of the AY [18]. In the Defensive Burying test, an anxiety/stress paradigm, Gutierrez-Mariscal et al. [23] showed that intracranial injection of TRH into AY prevents this stressinduced behavior in Wistar rats, but shock stress from an electrified probe reduced TRH activity in AY + PIR. The reduced TRH activity was shown by reduction of its mRNA and increased content (diminished release) 30 and 60 min after stress. The significant increases
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of Val-TRH levels at all times in AY in SD rats and of TRH at 2 h in the more stress-prone Lewis rats indicate peptide-specific inhibition of release of these peptides in the two strains. It is possible that this pharmacological regulation underlies the high frequency, deep brain stimulation (thought to inhibit the fear circuitry) of the basolateral nucleus of the AY which alleviates post-traumatic stress disorder symptoms in a rat model [32]. Neuroprotective ␣1A -adrenoreceptors have been reported in a subpopulation of CA1 interneurons of the HC [25]. Long-term depression (LTD) in HC “mediates the impairment of acute stressinduced spatial memory retrieval in rats” [64]. Furthermore, LTD is induced in rats by activation of ␣1 -noradrenergic receptors in HC but blocked by prazosin [56]. The many significant acute changes in TRH-like peptide levels in HC of both Lewis and SD rats induced by prazosin is consistent with the sensitivity of the HC to stressinduced pathology [45], and potential mediation of some of the therapeutic effects of prazosin by TRH and TRH-like peptides. The fewer prazosin-induced changes in Lewis rats suggest reduced sensitivity to this drug. Among the significant responses to a single ip injection of prazosin in SD rats was a fall in TRH and TRH-like peptide levels in MED, suggesting induced release. No evidence for prazosin-induced peptide release was observed in MED of Lewis rats. TRH and TRH-like peptides are contained within large dense core vesicles (LDCV) that consist of readily releasable LDCVs and reserve LDCVs that require more than 2 h to become activated for release [38]. Replenishment of depleted LDCV pools also requires more than 2 h. Conversely, a significant rise in peptide level at 2 h suggests inhibition of release. Intracisternal (ic) injection of TRH or a TRH analog, stimulates both pancreatic insulin release and hepatic glucose secretion [2]. TRH-induced insulin release was completely blocked by bilateral cervical vagotomy and the hyperglycemia was prevented by adrenalectomy [2]. A sympathetic-adrenal inhibitory tone on basal insulin and TRH [6,15,17,19] is maintained by the midbrain TRHcontaining neurons [2]. Prazosin blocks excitatory adrenoceptors in pancreatic ganglia [65] and the norepinephrine-inhibited release of glucagon by alpha cells within the islets of Langerhans [21,62]. Prazosin acutely stimulates both insulin release by the pancreatic -cells as well as glucose release by the liver ([1] and this study). The fall in pancreatic TRH content (Table 5) involves corelease with insulin (see Section 3.8) since TRH is localized exclusively in the islet -cells [50]) and responds to the same secretegogues as insulin [6]. Depletion of TRH and TRH-like peptides from the adrenals of SD rats after all doses of prazosin (Table 5) is consistent with a role for intra-adrenal release of these peptides in the regulation of CORT release by the adrenals [14]. The total TRH-IR contents of the adrenal cortex and medulla are almost equal [58]. As seen in Fig. 5, TRH predominates in the adrenal cortex while Trp-TRH is most abundant in the adrenal medulla. To summarize, comparisons of altered brain levels of TRH and TRH-like peptides following ip prazosin, a specific antagonist of ␣1 -adrenoceptors, in SD and Lewis rats, paralleled the relative abundance of NA and ␣1 -adrenergic receptors. The adrenals of SD rats are more responsive to prazosin-induced release of CORT than those of Lewis rats, a potential factor reducing the vulnerability of SD rats to stress-induced PTSD-like symptoms [11]. We conclude that modulation of noradrenergic neurotransmission by release of TRH and TRH-like peptides in specific CNS regions may play a role in symptomatic improvements in PTSD patients treated with prazosin.
Conflict of interest None.
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Rapid modulation of TRH and TRH-like peptide release in rat brain and peripheral tissues by prazosin Albert Sattin a,b,c,e , Albert Eugene Pekary b,d,f,∗ , James Blood b a
Psychiatry Services, VA Greater Los Angeles Healthcare System, United States Research Services, VA Greater Los Angeles Healthcare System, United States c Departments of Psychiatry & Biobehavioral Sciences, University of California, Los Angeles, CA 90073, United States d Center for Ulcer Research and Education, VA Greater Los Angeles Healthcare System, United States e Brain Research Institute, University of California, Los Angeles, CA 90073, United States f Department of Medicine, University of California, Los Angeles, CA 90073, United States b
a r t i c l e
i n f o
Article history: Received 13 April 2011 Received in revised form 11 June 2011 Accepted 13 June 2011 Available online 28 June 2011 Key words: Post-traumatic stress disorder Thyrotropin releasing hormone Limbic system
a b s t r a c t Hyperresponsiveness to norepinephrine contributes to post-traumatic stress disorder (PTSD). Prazosin, a brain-active blocker of ␣1 -adrenoceptors, originally used for the treatment of hypertension, has been reported to alleviate trauma nightmares, sleep disturbance and improve global clinical status in war veterans with PTSD. Thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH2 ) may play a role in the pathophysiology and treatment of neuropsychiatric disorders such as major depression, and PTSD (an anxiety disorder). To investigate whether TRH or TRH-like peptides (pGlu-X-Pro-NH2 , where “X” can be any amino acid residue) participate in the therapeutic effects of prazosin, male rats were injected with prazosin and these peptides then measured in brain and endocrine tissues. Prazosin stimulated TRH and TRH-like peptide release in those tissues with high ␣1 -adrenoceptor levels suggesting that these peptides may play a role in the therapeutic effects of prazosin. Published by Elsevier Inc.
1. Introduction A noradrenergic basis for the symptomatology of post-traumatic stress disorder (PTSD) was suggested by the significant elevation of CSF norepinephrine (NE) and direct correlation of individual levels with scores on the Clinician-Administered PTSD scale for 11 combat veterans (p = 0.004; [22]). This concept was further supported by results of clinical trials with the adrenolytic drugs, prazosin and clonidine both of which were effective in reducing or arresting the principal symptoms of PTSD, implying pharmacological specificity via reduction of ␣1 agonism in the CNS [by prazosin: 7, 9, 53, 59]. There is a single post-mortem study of locus coeruleus (LC) neuron counts in three US veterans with likely war-related PTSD showing no overlap with the counts of NE neurons from 4 controls and a 47% reduction in mean count suggesting neurodegeneration and compensatory hypersecretion of NE [8]. PTSD is diagnostically distinct from Major Depression (MDD) yet there is significant symptomatic overlap as well as clinical co-morbidity [10]. As in PTSD, elevated NE has also been documented in CSF of melancholic Major Depressive
∗ Corresponding author at: VA Greater Los Angeles Healthcare System, Bldg. 114, Rm. 229, 11301 Wilshire Blvd., Los Angeles, CA 90073, United States. Tel.: +1 310 268 4430; fax: +1 310 441 1702. E-mail addresses: [email protected], [email protected] (A.E. Pekary). 0196-9781/$ – see front matter. Published by Elsevier Inc. doi:10.1016/j.peptides.2011.06.012
Disorder (MDD) [63]. Post-mortem NE neuron counts in LC were also reduced in depressive suicides (6 vs. 11 controls; [3]). But both the elevations of NE in CSF of patients and the reductions of NE neuron counts post-mortem appear to be more extreme in PTSD than in MDD from these data: 41% increase vs. 33% increase in CSF; 47% decrease vs. 23% decrease in LC. A noteworthy pathophysiological difference between these two diseases is the normality of circulating cortisol levels in PTSD vs. its sustained elevation in melancholic MDD but in CSF, cortisol was elevated in both PTSD and MDD [4,63]. A role for glutamate is implicated in PTSD, as well as for other disorders of anxiety and affect [24,36]. Preliminary evidence suggests that the anti-glutamatergic agents lamotrogine and memantine reduce PTSD symptoms [5]. Thyrotropin-releasing hormone (TRH) is an endogenous antidepressant, neuroprotective, anti-epileptic tripeptide that is one of many co-transmitters within glutamatergic neurons [26,31,33] which are involved in the pathophysiology of many neuropsychiatric disorders (reviewed by Sattin [54]). These and other functions of TRH should be distinguished from the traditional neuroendocrine role of hypothalamic TRH. Along with glutamate, TRH is releasable from HC ex vivo [30] and it has neuroprotective effects on glutamatoceptive neurons in vitro [29]. In contrast to the hypothalamus where TRH itself predominates, suprahypothalamic TRH (pGlu-His-NH2 ) is one among many TRHlike peptides with the general structure of pGlu-X-Pro-NH2 where
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Table 1 Control and post-prazosin injection serum values of corticosterone, free T4, total T3 and glucose. *(p < 0.05); **(p < 0.025); ***(p < 0.01) by one-way ANOVA vs. control (# higher than mean for Lewis rats by 2-tailed non-paired t test). Rat strain group
Corticosterone ng/ml
Total T3 ng/dl
Free T4 ng/dl
Glucose mg/dl
SD Control 2h 4h 6h
183 540 539 408
± ± ± ±
65# 36* 74* 175*
2.13 1.93 1.57 1.21
± ± ± ±
0.64 0.09 0.26 0.15
54 42 41 31
± ± ± ±
12 4 17 4*
116 381 246 146
± ± ± ±
7 90*** 70* 36
Lewis Control 2h 4h 6h
36 284 332 301
± ± ± ±
38 119* 32** 97*
2.29 1.61 1.57 1.27
± ± ± ±
0.14 0.30* 0.10** 0.18***
43 40 28 24
± ± ± ±
7 7 4* 1**
110 219 220 135
± ± ± ±
9 75* 40* 14
Table 2 Effects of prazosin (single ip injection) on HPLC peak areas corresponding to TRH and TRH-like peptide levels in various brain regions of male Sprague–Dawley rats involved in regulation of mood and behavior. Glu-TRH Nucleus accumbens 0.60 2h 1.47 4h 1.36 6h
Peak 2
TRH
Val-TRH
Tyr-TRH
Leu-TRH
Phe-TRH
Trp-TRH
0.62 1.19 ↑2.57**
0.52 2.47 3.46
0.13 1.35 0.97
0.46 1.97 1.57
1.00 3.09 2.18
0.12 2.61 0.08
0.45 4.15 4.03
1.18 0.46 1.32
2.29 1.76 1.98
0.92 0.76 ↑1.66**
↑3.16* ↑3.58* ↑2.55*
1.88 1.28 0.97
2.50 2.91 5.32
1.64 1.96 1.47
0.31 0.29 0.53
Piriform cortex 0.60 2h 4h 0.99 0.38 6h
0.70 0.50 ↓0.31
0.74 1.34 0.47
↑3.97* 1.55 0.52
2.12 0.96 0.42
1.82 0.70 0.50
↑3.05* 0.75 0.51
0.22 0.78 0.26
Frontal cortex 2h 0.64 4h 0.61 1.02 6h
2.08 5.53 2.43
1.86 2.19 2.19
2.44 ↑7.00** 1.85
0.96 ↑1.58* ↑1.89*
0.98 ↑2.89* 1.89
1.07 ↑4.25* ↑3.47*
1.24 0.80 ↑2.74*
Hippocampus 1.31 2h 0.64 4h 1.68 6h
1.42 1.51 2.34
1.01 1.42 2.82
↑2.18* ↑2.27* ↑2.16*
4.61 2.42 1.64
2.78 1.45 1.11
1.27 ↑4.75* 2.12
↓0.43* ↓0.26** ↓0.44*
Anterior cingulate ↑2.71* 2h ↓0.17* 4h 0.46 6h
0.62 ↓0.34* 0.89
1.60 0.20 2.30
1.85 0.12 0.37
↑3.76* 0.25 0.66
3.19 0.46 1.25
0.68 0.62 0.11
2.26 0.27 ↑4.76*
Posterior cingulate 0.88 2h 0.73 4h 0.60 6h
1.10 1.36 1.11
0.83 ↑3.13* 2.03
1.32 1.14 1.20
1.78 1.13 1.03
0.80 1.15 1.78
0.58 0.96 0.25
0.62 0.38 0.83
Entorhinal cortex 0.19 2h 2.07 4h 2.06 6h
0.50 1.08 ↑1.55*
0.47 1.02 ↑2.39*
0.65 2.02 ↑2.82*
1.63 ↑5.96* ↑5.37*
0.50 ↑3.97* 1.91
1.39 5.20 6.87
1.94 1.12 1.42
Striatum 2h 4h 6h
1.35 2.75 ↑3.82*
1.14 1.65 ↑4.08**
0.73 1.52 2.22
0.62 1.89 ↑2.49*
2.19 2.54 ↑4.84*
Amygdala 2h 4h 6h
0.85 0.86 1.78
0.80 1.50 ↑5.11***
0.61 1.40 1.17
Cerebellum 2h 0.87 ↓0.36** 4h ↓0.32** 6h
↓0.30*** ↓0.22*** ↓0.17**
1.82 2.22 1.78
0.96 0.70 0.84
0.49 0.87 0.46
4.08 1.36 1.53
0.87 0.81 0.79
0.87 0.46 0.56
Medulla oblongata 2h 0.38 1.13 4h – 6h
↓0.33* ↑1.98* 0.49
0.39 1.52 0.75
0.46 ↑3.28* 0.62
0.50 0.83 ↓0.21*
0.54 1.67 0.30
0.49 0.94 1.94
↓0.08* 1.30 ↓0.04*
Results are peak area divided by corresponding peak area in controls. *p < 0.05; **p < 0.025; ***p < 0.025 by one way ANOVA using post hoc Scheffe contrasts vs. the control group [47]. (–) Fractions lost during HPLC.
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Table 3 Effects of prazosin (single ip injection) on HPLC peak areas corresponding to TRH and TRH-like peptide levels in various brain regions of male Lewis rats involved in regulation of mood and behavior. Glu-TRH
TRH
Val-TRH
Tyr-TRH
0.66 0.90 1.39
1.12 0.95 ↑1.97*
1.37 ↓0.46* ↓0.34*
1.23 ↑3.00* 0.95
1.73 0.55 2.38
2.09 2.13 1.98
1.01 0.75 1.14
1.88 1.51 1.65
1.34 0.95 1.03
↑1.33* 0.82 ↑1.35*
1.37 2.13 2.74
0.59 0.57 0.75
0.79 1.34 0.74
1.66 0.73 1.19
1.40 0.66 1.34
Piriform cortex 0.64 2h 4h 0.54 6h 0.27
1.13 0.76 0.66
0.97 0.58 0.99
1.12 0.78 ↓0.24*
↑4.72* 0.89 0.22
1.88 1.15 1.21
1.00 0.52 0.45
0.68 0.46 0.60
Frontal cortex 2h 4h 6h
0.64 0.79 0.78
1.63 2.19 2.74
2.07 ↑5.63* 2.71
1.09 2.76 ↑7.04**
0.92 1.35 1.16
1.18 0.85 ↑2.23*
1.06 0.71 ↑3.57*
1.13 0.97 1.47
Hippocampus 2h 4h 6h
2.22 0.90 0.45
0.54 0.43 ↓0.25*
0.95 0.35 0.25
1.43 1.12 0.43
1.66 0.91 0.67
0.76 0.37 0.55
↓0.25* ↓0.22* ↓0.09**
↑2.98** 1.58 0.59
Anterior cingulate 1.15 2h 4h 1.12 ↑1.89* 6h
0.88 1.16 ↑3.06*
1.56 ↑6.03* 2.62
0.38 0.52 1.03
2.04 0.96 2.09
0.25 0.47 0.41
1.02 1.66 1.08
0.55 0.67 0.63
Posterior cingulate 1.26 2h 4h 0.87 6h 1.16
1.00 0.56 2.58
0.91 0.81 0.58
0.62 0.71 0.59
1.10 1.02 0.49
1.45 0.81 1.02
↑14.36** 1.15 0.78
2.04 1.07 1.22
Entorhinal cortex 0.61 2h 4h 1.04 1.30 6h
↓0.51* 0.61 1.04
1.01 ↑2.35* 2.24
1.54 2.43 ↑3.16*
0.91 1.12 ↑3.10*
1.12 1.75 1.69
1.22 1.65 3.73
0.93 0.94 1.75
1.17 0.84 1.03
1.14 0.65 0.97
1.04 0.81 1.14
0.91 0.52 1.74
1.02 0.84 2.14
1.19 0.71 0.87
1.33 1.13 1.36
0.64 ↓0.53* 0.84
1.25 ↓0.49* ↓0.47*
0.83 0.80 ↓0.49*
1.59 0.57 0.63
1.47 ↓0.34* 0.51
1.44 1.51 0.50
2.10 1.49 0.73
1.62 1.31 0.54
1.09 1.09 0.68
1.59 1.85 ↑2.90*
1.78 1.70 1.51
1.00 1.61 ↑2.79*
1.69 3.81 3.48
1.94 5.50 3.16
1.77 2.83 1.37
1.22 1.80 2.27
Nucleus accumbens 1.25 2h 1.09 4h 0.82 6h Amygdala 2h 4h 6h
Striatum 2h 4h 6h Cerebellum 2h 4h 6h
Medulla oblongata 1.60 2h 1.99 4h 1.41 6h
Peak 2
Leu-TRH
Phe-TRH
Trp-TRH
*p < 0.05; **p < 0.025; ***p < 0.025 by one way ANOVA using post hoc Scheffe contrasts vs. the control group [47].
X = any amino acid. This also occurs in many peripheral tissues. Phe-TRH (X = Phe) exhibits marked circadian rythmicity [47]. Neuroprotective effects of Glu-TRH were up to fourfold greater than TRH [29]. Both TRH and Glu-TRH are augmented in brain after electroconvulsive seizures [54]; TRH confirmed by Newton et al. [37]. Psychopharmacological agents and the photoperiod [41,46,47,50], thyroid hormones, glucocorticoids, GSK-3, lipopolysaccharide, leptin, and ghrelin also modulate these tripeptides in brain and peripheral tissues. Leptin and ghrelin evoke the most rapid alterations of these peptides in brain and periphery suggesting their involvement in downstream effects, including their antidepressant effects [43,48]. In the present study we compare the effect of prazosin on the expression of TRH and TRH-like peptides (pGlu-X-Pro-NH2 ) in the brain and peripheral tissues of young adult male Lewis and SD rats, two strains that have been reported to have high and moderate Extreme Behavioral Responses (EBR) to predator scent, respectively, an animal model of PTSD [11,12].
2. Materials and methods 2.1. Animals Male Sprague–Dawley (SD) and Lewis rats (Harlan, Indianapolis, IN) were used for all experiments. These animals were group housed (4 animals per cage), maintained with standard Purina rodent chow #5001 and water ad libitum during a standard one week initial quarantine in a controlled temperature and humidity environment; lights on: 6 a.m. to 6 p.m. All animals were weighed on the day of receipt and on the morning of each experiment. Initial and final body weights did not differ between experimental groups. Research was approved by the VA Greater Los Angeles Healthcare System Animal Care and Use Committee and conducted in compliance with the Animal Welfare Act and the federal statutes and regulations related to animals and experiments involving animals and adhere to principles stated in the Guide for the Care and use of Laboratory Animals, NRC Publication, 1996 edition. All efforts
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Table 4 Concentration-dependent effects (0, 0.01, 0.1 and 1.0 × 3.9 mg/kg) 4 h after a single ip injection of prazosin on TRH and TRH-like peptide levels in various brain regions involved in mood regulation of male SD rats. Glu-TRH Nucleus accumbens 0.51 0.01 0.86 0.1 0.66 1.0
Peak 2
TRH
Val-TRH
Tyr-TRH
Leu-TRH
Phe-TRH
Trp-TRH
0.87 ↑2.27** 0.69
1.22 1.73 1.84
1.40 1.47 0.84
0.91 ↑2.35* 0.56
0.27 0.96 1.18
0.40 ↑3.41** 1.19
1.06 1.05 2.02
Amygdala 0.01 0.1 1.0
0.71 0.85 0.77
0.47 0.74 0.56
1.30 0.99 0.99
0.45 0.58 0.54
0.48 0.62 0.44
0.52 0.79 0.76
0.62 1.00 1.21
0.92 1.08 0.54
Piriform cortex 0.01 0.1 1.0
0.74 0.45 0.36
1.81 0.55 0.64
1.63 0.98 0.84
1.19 ↓0.31* 0.52
1.57 0.65 0.56
1.66 0.50 0.73
1.52 0.79 0.54
1.34 0.57 0.72
Frontal cortex 1.25 0.01 1.20 0.1 ↓0.32* 1.0
1.73 1.77 0.76
2.15 0.59 0.40
1.62 0.40 0.24
↑1.91** ↓0.49* ↓0.51*
↑1.94* 0.43 0.32
1.39 0.59 0.42
1.31 0.44 ↓0.27*
Hippocampus 0.01 0.1 1.0
0.94 0.45 0.48
2.42 1.07 1.8
1.45 0.66 0.68
1.80 0.51 0.70
0.98 0.40 0.39
0.55 ↓0.16* 0.22
0.96 ↓0.24* 0.38
Anterior cingulate ↓0.38* 0.01 0.1 0.93 ↓0.03* 1.0
1.26 0.57 0.49
1.02 0.50 0.32
0.79 ↓0.20* ↓0.15*
0.96 ↓0.32* ↓0.19*
0.53 0.30 0.20
1.35 0.38 0.28
1.41 0.73 0.64
Posterior cingulate 0.98 0.01 0.1 0.27 1.0 0.92
0.78 1.16 2.10
0.62 1.17 1.30
0.54 0.93 1.30
0.38 1.53 1.45
0.48 0.72 1.03
1.37 1.53 1.98
0.84 1.55 1.58
Entorhinal cortex 0.76 0.01 0.1 0.80 0.96 1.0
↓0.61* ↓0.41* 0.85
1.28 0.51 1.40
1.41 0.60 0.96
1.54 0.58 1.07
0.85 0.50 0.89
1.00 1.10 1.02
1.49 1.10 2.21
0.81 0.59 0.77
0.82 0.55 0.43
↓0.54* ↓0.18** ↓0.34**
1.16 0.50 0.48
↓0.29* ↓0.24* ↓0.26*
↓0.56* ↓0.30** ↓0.23**
↑2.98** 1.70 ↑1.96*
↑1.97* 1.36 ↑2.11*
↑3.89* 1.74 ↑3.35*
1.39 0.63 1.28
2.46 2.11 2.25
1.79 1.22 2.21
↑3.44* 1.73 ↑3.60*
2.26 1.61 2.19
↓0.33** 0.76 ↓0.00***
↓0.30* 0.37 ↓0.36*
↓0.27* 0.55 ↓0.49*
0.61 0.87 0.51
↓0.18* 0.41 0.27
↓0.05* ↓0.27* ↓0.09*
↓0.13* ↓0.29* ↓0.19*
Striatum 0.01 0.1 1.0 Cerebellum 0.01 0.1 1.0
0.64 0.47 0.43
Medulla oblongata 0.68 0.01 0.87 0.1 0.66 1.0
↓0.64** ↓0.14*** ↓0.08***
0.46 ↓0.25** ↓0.16**
*p < 0.05; **p < 0.025 by one way ANOVA using post hoc Scheffe contrasts vs. the control group as previously described [47].
have been made to minimize the number of animals used and their suffering. All animals were transferred from the Veterinary Medical Unit to the laboratory 12 h before the start of experiments to minimize the stress of a novel environment.
such that decapitation could occur at 20 min intervals between 9 a.m. and 11 a.m. to minimize the effect of the circadian rhythm on TRH and TRH-like peptide levels in rat brain [47]. 2.3. Pharmacokinetic properties of prazosin in rat
2.2. Ip vs. oral administration of prazosin Prazosin was administered ip rather than orally because firstpass metabolism of prazosin is partially responsible for its low oral bioavailability. Hepatic metabolism of prazosin accounts for more than 95% of its elimination, almost all of the metabolites are excreted in bile without re-entry into the systemic circulation [28]. The plasma clearance times for prazosin following oral and ip administration in normal human subjects are identical, 2.9 ± 0.56 and 2.9 ± 0.75 h, respectively [28]. Animals were injected at times
The relative amount of the injected dose of prazosin taken up by tissues is liver > kidneys > plasma = lungs > heart > spleen > brain. The percentage in brain is 4%. The clearance time (t1/2 ) of 44 min in rats does not differ significantly among tissues or plasma [16]. 2.4. Time-dependent effects of prazosin on TRH and TRH-like peptide levels in brain of SD rats Male SD rats, weighing 346 ± 17 g, were either uninjected (control group), or received a single 1.0 ml ip injection of 1.3 mg
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Table 5 Concentration-dependent effects (0, 0.01, 0.1 and 1.0 × 3.9 mg/kg) 4 h after a single ip injection of prazosin on TRH and TRH-like peptide levels in adrenal, pancreatic and reproductive tissues of male SD rats. Glu-TRH
Peak 2
Adrenal 0.01 0.1 1.0
TRH
Val-TRH
Tyr-TRH
Leu-TRH
Phe-TRH
Trp-TRH
↓0.24* ↓0.12** ↓0.30*
↓0.13** ↓0.47* ↓0.43*
0.63 0.73 0.52
0.63 0.66 0.66
1.04 1.76 0.53
0.9 1.42 0.67
0.91 1.40 0.47
1.33 ↑2.27* ↓0.41*
Pancreas 0.01 0.1 1.0
↓0.24** 0.78 ↓0.11**
0.51 ↓0.35* 0.68
0.52 1.10 1.02
0.81 0.78 0.8
↓0.41* 0.66 0.74
0.53 1.37 0.83
0.53 0.88 ↓0.28*
0.52 1.17 0.80
Testis 0.01 0.1 1.0
1.95 ↑2.62* ↑9.09***
0.41* 1.63 ↑2.49*
0.56 1.76 1.98
0.50 1.84 ↑4.12**
↓0.42* 1.43 1.60
↓0.36* 1.20 1.50
↓0.19** 1.14 0.98
↓0.29* 1.92 1.71
Epididymis 0.01 0.1 1.0
0.55 ↓0.40* ↓0.42*
↓0.19** ↓0.14** ↓0.21**
0.74 ↓0.27* ↓0.34*
0.50 ↓0.29* ↓0.36*
0.58 ↓0.30* ↓0.37*
↓0.34* ↓0.15** ↓0.29*
↓0.34* ↓0.15** ↓0.22**
↓0.31* ↓0.31* ↓0.23**
Prostate 0.01 0.1 1.0
↓0.32* ↓0.06*** ↓0.03***
↓0.41* ↓0.32* ↓0.34*
↓0.30* ↓0.49* ↓0.48*
↓0.38* ↓.17** ↓0.13**
↓0.37* ↓0.39* ↓0.24**
↓0.19** ↓0.17** ↓0.16**
↓0.25* ↓0.16** ↓0.12***
↓0.21** ↓0.36* ↓0.23**
*p < 0.05; **p < 0.025 by one way ANOVA using post hoc Scheffe contrasts vs. the control group as previously described [47].
Fig. 1. Concentration-dependent effects 4 h after a single ip injection of prazosin on TRH and TRH-like peptide levels in striatum of male SD rats. Note the progressive fall in TRH, Tyr-TRH, Leu-TRH, Phe-TRH and Trp-TRH levels with increasing prazosin concentration.
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Fig. 2. Concentration-dependent effects 4 h after a single ip injection of prazosin on TRH and TRH-like peptide levels in medulla oblongata of male SD rats. TRH and Peak 2, Val-TRH, Leu-TRH, Phe-TRH and Trp-TRH levels were significantly suppressed by 0.01× the maximum prazosin dose.
prazosin (3.8 mg/kg body weight) (Sigma, St. Louis, MO)/ml of 50% ethanol + 50% dimethyl sulfoxide 2, 4 or 6 h (n = 4 for each time point) before decapitation.
2.5. Time-dependent effects of prazosin on TRH and TRH-like peptide levels in brain of Lewis rats Male Lewis rats, weighing 258 ± 13 g, were either uninjected (control group), or received a single 0.7 ml ip injection of 1.5 mg prazosin (4.1 mg/kg body weight) (Sigma, St. Louis, MO)/ml of 50% ethanol + 50% dimethyl sulfoxide 2, 4 or 6 h (n = 4 for each time point) before decapitation.
2.7. Dissection of rat brain, pancreas, adrenals and reproductive organs After decapitation, nucleus accumbens (NA), amygdala (AY), frontal cortex (FCX), cerebellum (CBL), medulla oblongata (MED), anterior cingulate (ACNG), posterior cingulate (PCNG), striatum (STR), piriform cortex (PIR), hippocampus (HC), entorhinal cortex (ENT), pancreas, prostate, epididymis, testes and adrenals were hand dissected, weighed rapidly, and then extracted as previously described [41,44–51,55]. In a separate experiment, 4 rats were decapitated, the adrenals removed, and each adrenal cortex and medulla separated, with the aid of a dissecting microscope, and extracted as described above.
2.6. Concentration-dependent effects of prazosin on TRH and TRH-like peptide levels in brain and peripheral tissues of SD rats
2.8. Serum hormone assays
Male SD rats, weighing 257 ± 64 g, were injected with 0.5 ml of 50% DMSO + 50% ethanol containing 0, 0.01, 0.1 or 1.0 × 2.0 mg prazosin/ml (mean of 3.9 mg/kg prazosin at the highest dose) followed 4 h later by decapitation. The lowest dose corresponds to the typical 2–5 mg oral dose in patients.
Serum glucose was measured with the OneTouch Ultra Blood Glucose Monitoring System, Life Scan, Milpitas, CA. Serum corticosterone (CORT), free T4 , and total T3 were measured with the following commercial RIA kits: CORT (MP Biomedical, Aurora, OH), free T4 and total T3 (DPC Coat-A-Count, Los Angeles, CA). Rat insulin
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Fig. 3. Concentration-dependent effects 4 h after a single ip injection of prazosin on TRH and TRH-like peptide levels in epididymis of male SD rats. TRH and all TRH-like peptide levels fell progressively with increasing prazosin concentration.
and rat leptin RIA kits were purchased from Millipore, St. Charles, MO.
3. Results 3.1. Serum values of corticosterone, free T4 , total T3 , and glucose for time-dependent effects in SD rats
2.9. HPLC and RIA procedures, HPLC peak identification and quantitation HPLC and RIA procedures, peak identification, and quantitation by co-chromatography with synthetic TRH and TRH-like peptides, relative potency analysis of multiple antibodies to TRH and TRHlike peptides, mass spectrometry and resolution of overlapping peaks by least squares fitting of a 2-Gaussian statistical model have been previously reported in detail [41,44–51,55]. “Peak 2” is a mixture of at least 2 unidentified TRH-like peptides.
2.10. Statistical analysis Statistical comparisons were made with the aid of Statview (Abacus Concepts, Inc., Berkeley, CA), a statistical software package for the Macintosh computer. All multi-group comparisons were carried out by one way analysis of variance using post hoc Scheffe contrasts with the control group [44,47,48].
Serum corticosterone levels in SD rats increased 3-fold at 2 h followed by a progressive decline to a 2.2-fold increase at 6 h post injection. Total T3 levels decline progressively to 43% of the control value by 6 h due, in part, to the blockade of sympathetic nervous system stimulation of the type 2 deiodinase in brain and brown fat which converts T4 to T3 [52,57]. Glucose rose 3.3-fold at 2 h followed by a progressive decline to control values at 6 h. This rise can be attributed to a decrease in glucose uptake in muscle [27] following ␣1 -adrenoceptor blockade by prazosin and stimulation of corticosterone release [49] (see Table 1 and Section 3.8). 3.2. Serum values of corticosterone, free T4 , total T3 , and glucose for time-dependent effects in Lewis rats Serum corticosterone levels in Lewis rats were increased: 8(2 h), 9.2- (4 h) and 8.4-fold (6 h). Serum free T4 declined 30% (2 h), 31% (4 h) and 45% (6 h) while serum total T3 fell 35% (4 h) and 44% (6 h) due to increased glucocorticoid inhibition of pituitary TSH
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Fig. 4. Concentration-dependent effects 4 h after a single ip injection of prazosin on TRH and TRH-like peptide levels in prostate of male SD rats. TRH and all TRH-like peptide levels fell progressively with increasing prazosin concentration.
release decreased sensitivity to HY TRH release [61], and decreased noradrenergic stimulated conversion of T4 to T3 [57]. However, the absolute increase in corticosterone and glucose in Lewis rats was less than for the SD rats, despite a slightly higher dose of prazosin (see Table 1 and Section 3.8).
3.3. Overview of HPLC results for time-dependent effects in SD and Lewis rats The acute effect of a single ip injection of prazosin on levels of TRH and TRH-like peptides in brain and peripheral tissues was, in general, heterogeneous. Depending on the particular peptide and tissue or brain region, responses in peptide levels ranged from (a) no effect, (b) a rapid and sustained rise, (c) a rapid rise followed by a gradual decline toward the control value, (d) a rapid and sustained decline, or a rapid decline followed by a recovery to the control value. A significant decrease in peptide level at 2 h is consistent with acceleration of release [38]. Rapid change in TRH and TRH-like peptide levels was not a nonspecific response to the acute stress of the ip injections administered at 2, 4 and 6 h before decapitation. For example, ip injection of 14.1 mg/kg corticosterone resulted in a serum level of 27,806 ng/ml corticosterone at 2 h. Nevertheless, there were no
significant changes in TRH or TRH-like peptide levels in brain or peripheral tissues at 2 h compared to uninjected controls [49]. 3.4. Time-dependent effects of prazosin at 2 h in brain of SD rats Peak 2 and Trp-TRH levels in MED decreased 67% and 92%, respectively, by 2 h consistent with prazosin-induced release of these peptides (see Table 2). We abbreviate this result as: MED (2↓). Significant changes in TRH and TRH-like peptide levels for other brain regions were: ACNG (2↑), PIR (2↑), HC (1↓, 1↑), CBL (1↓), AY (1↑), for a total of 10 (Table 2). 3.5. Time-dependent effects of prazosin at 4 and 6 h in brain of SD rats Significant changes were: FCX (7↑), ENT (6↑), STR (5↑), HC (2↓, 3↑), MED (2↓, 2↑), AY (3↑), PIR (1↓), NA (1↑), PCNG (1↑), for a total of 33 (Table 2). 3.6. Time-dependent effect of prazosin at 2 h in brain of Lewis rats HC (1↓, 1↑), AY (1↑), PIR (1↑), PCNG (1↑) and ENT (1↓) changes totaled 6 (Table 3).
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Fig. 5. Comparison of the TRH and TRH-like peptide levels in the adrenal cortex and medulla of male SD rats. Note, for example, that TRH is 65% higher and Trp-TRH is 57% lower in the cortex compared to the medulla.
3.7. Time-dependent effect of prazosin at 4 and 6 h in Lewis rat brain
3.10. Concentration-dependent effects of prazosin in reproductive and pancreatic tissues
CBL (4↓), FCX (4↑), NA (2↓, 2↑), HC (3↓), ACNG (3↑), ENT (3↑), MED (2↑), AY (1↑), PIR (1↓), STR (1↓), totaled 26 (Table 3).
Prazosin at 0.01, 0.1 and 1.0 times the maximum dose had a profound suppressive effect on the levels of TRH and TRH-like peptide levels (increased release) in prostate and epididymis of SD rats 4 h after ip injection of prazosin (Table 5; Figs. 3 and 4).
3.8. Serum values for glucose, insulin, leptin, free T3, free T4, corticosterone, testosterone, for concentration-dependent effects
4. Discussion Serum leptin, free T3 , free T4 , corticosterone and testosterone levels were not significantly changed by increasing dose of prazosin vs. the vehicle (control) 4 h after injection (results not shown). The corresponding serum glucose values (mean ± SD, mg/dl) were: 65 ± 8 (control); 146 ± 40 (0.01× dose, n.s.); 132 ± 20 (0.1× dose, n.s.); 255 ± 130, (1× dose, p < 0.05). Serum insulin (mean ± SD, ng/ml): 0.05 ± 0.00 (control); 0.05 ± 0.00 (0.01× dose, n.s.); 0.08 ± 0.05 (0.1× dose, n.s.); 0.63 ± 0.64, (1× dose, p < 0.05). 3.9. Overview of concentration-dependent HPLC results in SD brain after ip prazosin Because of the limited solubility of prazosin in water and ethanol, DMSO is the solvent most commonly used for its in vivo administration. DMSO, however, has been reported to have a highly inhibitory effect on acetylcholinesterase at all concentrations tested [39]. As a result, the time-dependent effects of a single ip injection of prazosin in DMSO should be a combination of the prazosin and DMSO effects. Rather than performing a separate, time-dependent, control experiment involving the injection of DMSO alone, a concentration-dependent study of the prazosin effects at 4 h was carried out. This time point provided more significant responses than 2 h. Measurement of the TRH and TRH-like peptide responses to 0, 1, 10 and 100% of the time-dependent dose for prazosin should identify those tissues most sensitive to the effects of prazosin and properly control for any DMSO effects. Even though all of the concentration-dependent effects were obtained at 4 h after ip injection of prazosin it is possible to attribute changes in peptide levels at this time to altered release/degradation since ␣1 -adrenoreceptors regulate TRH secretion but not proTRH biosynthesis and processing [38] (see Tables 4 and 5; Figs. 1 and 2). The significant changes in TRH and TRH-like peptide levels for all prazosin concentrations tested were: STR (14↓); MED (13↓); CBL (8↑); ACNG (6↓); FCX (5↓,1↑); NA (3↑); HC (2↓); ENT (2↓); PIR (1↓); PCNG (0).
The most striking observation in the present study was the nearly quantitative release/clearance of all TRH and TRH-like peptides from the prostate and epididymis at even a very low dose of prazosin 4 h after injection. Contraction [60] and growth [34] of the prostate and epididymis are activated by noradrenergic sympathetic innervation of the membrane-bound and intracellular ␣1 -adrenoceptors found throughout the ejaculatory system of rats and man [13]. Prazosin and other ␣1 -adrenoceptor inhibitors are used for the treatment of the urologic complications of benign prostatic hypertrophy [13]. These particular adrenoreceptors are also responsible for stimulating the release of TRH from the hypothalamus in response to cold, and inhibiting its release in response to stress, but they do not modulate its synthesis, as is the case for the -adrenoceptors [38]. The norepinephrine concentration in the rat reproductive system, which is comparable to that of cortex and other extrahypothalamic brain regions [40] appears to provide an inhibitory tone for TRH and TRH-like peptide release which is reversed by ␣1 -adrenoceptor inhibitors such as prazosin. The total TRH immunoreactivity of the rat prostate is comparable to that of the rat hypothalamus [42]. The TRH-like peptides Glu-TRH and Leu-TRH were elevated 25- and 22-fold, respectively, in the epididymis 2 h after injection of escitalopram, a highly selective serotonin reuptake inhibitor [55]. Glu-TRH is a fertilization promoting peptide [20]. The present results and previous studies [35,50] suggest that TRH and TRH-like peptides participate in the regulation of reproductive functions by neurotransmitters such as norepinephrine and serotonin. Prazosin blocks the ACTH and CORT responses to the stimulation of the central nucleus of the AY [18]. In the Defensive Burying test, an anxiety/stress paradigm, Gutierrez-Mariscal et al. [23] showed that intracranial injection of TRH into AY prevents this stressinduced behavior in Wistar rats, but shock stress from an electrified probe reduced TRH activity in AY + PIR. The reduced TRH activity was shown by reduction of its mRNA and increased content (diminished release) 30 and 60 min after stress. The significant increases
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of Val-TRH levels at all times in AY in SD rats and of TRH at 2 h in the more stress-prone Lewis rats indicate peptide-specific inhibition of release of these peptides in the two strains. It is possible that this pharmacological regulation underlies the high frequency, deep brain stimulation (thought to inhibit the fear circuitry) of the basolateral nucleus of the AY which alleviates post-traumatic stress disorder symptoms in a rat model [32]. Neuroprotective ␣1A -adrenoreceptors have been reported in a subpopulation of CA1 interneurons of the HC [25]. Long-term depression (LTD) in HC “mediates the impairment of acute stressinduced spatial memory retrieval in rats” [64]. Furthermore, LTD is induced in rats by activation of ␣1 -noradrenergic receptors in HC but blocked by prazosin [56]. The many significant acute changes in TRH-like peptide levels in HC of both Lewis and SD rats induced by prazosin is consistent with the sensitivity of the HC to stressinduced pathology [45], and potential mediation of some of the therapeutic effects of prazosin by TRH and TRH-like peptides. The fewer prazosin-induced changes in Lewis rats suggest reduced sensitivity to this drug. Among the significant responses to a single ip injection of prazosin in SD rats was a fall in TRH and TRH-like peptide levels in MED, suggesting induced release. No evidence for prazosin-induced peptide release was observed in MED of Lewis rats. TRH and TRH-like peptides are contained within large dense core vesicles (LDCV) that consist of readily releasable LDCVs and reserve LDCVs that require more than 2 h to become activated for release [38]. Replenishment of depleted LDCV pools also requires more than 2 h. Conversely, a significant rise in peptide level at 2 h suggests inhibition of release. Intracisternal (ic) injection of TRH or a TRH analog, stimulates both pancreatic insulin release and hepatic glucose secretion [2]. TRH-induced insulin release was completely blocked by bilateral cervical vagotomy and the hyperglycemia was prevented by adrenalectomy [2]. A sympathetic-adrenal inhibitory tone on basal insulin and TRH [6,15,17,19] is maintained by the midbrain TRHcontaining neurons [2]. Prazosin blocks excitatory adrenoceptors in pancreatic ganglia [65] and the norepinephrine-inhibited release of glucagon by alpha cells within the islets of Langerhans [21,62]. Prazosin acutely stimulates both insulin release by the pancreatic -cells as well as glucose release by the liver ([1] and this study). The fall in pancreatic TRH content (Table 5) involves corelease with insulin (see Section 3.8) since TRH is localized exclusively in the islet -cells [50]) and responds to the same secretegogues as insulin [6]. Depletion of TRH and TRH-like peptides from the adrenals of SD rats after all doses of prazosin (Table 5) is consistent with a role for intra-adrenal release of these peptides in the regulation of CORT release by the adrenals [14]. The total TRH-IR contents of the adrenal cortex and medulla are almost equal [58]. As seen in Fig. 5, TRH predominates in the adrenal cortex while Trp-TRH is most abundant in the adrenal medulla. To summarize, comparisons of altered brain levels of TRH and TRH-like peptides following ip prazosin, a specific antagonist of ␣1 -adrenoceptors, in SD and Lewis rats, paralleled the relative abundance of NA and ␣1 -adrenergic receptors. The adrenals of SD rats are more responsive to prazosin-induced release of CORT than those of Lewis rats, a potential factor reducing the vulnerability of SD rats to stress-induced PTSD-like symptoms [11]. We conclude that modulation of noradrenergic neurotransmission by release of TRH and TRH-like peptides in specific CNS regions may play a role in symptomatic improvements in PTSD patients treated with prazosin.
Conflict of interest None.
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