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WWW. M ED S CI M ONIT .COM Hypothesis Relaxation: Molecular and physiological significance Authors’ Contribution: A Study Design B Data Collection C Statistical Analysis D Data Interpretation E Manuscript Preparation F Literature Search G Funds Collection George B. Stefano1 DEG, Gregory L. Fricchione2 EF, Tobias Esch3 EF 1 SE Received: 2006.06.19 Accepted: 2005.07.03 Published: 2006.09.01 HY Neuroscience Research Institute, State University of New York College at Old Westbury, NY, U.S.A. Department of Psychiatry, Massachusetts General Hospital, Boston, MA, U.S.A. 3 Division of Integrative Health Promotion, Coburg University of Applied Sciences, Coburg, Germany 2 R SO O N N A LY L U Source of support: Supported in part by grants MH 47392 and DA 09010 (GBS) Summary There appears to be a molecular process for relaxation. Given this, we attempt to demonstrate this phenomenon based on established molecular and physiological processes in light of our current understanding of central and peripheral nervous system mechanisms. Central to our hypothesis is the significance of norepinephrine, nitric oxide, dopamine and morphine signaling both in the central and peripheral nervous system. We find that nitric oxide and morphine control catecholamine processes on many levels, including synthesis, release and actions. We conclude that enough scientific information exists to support these phenomena as actual physical processes that can be harnessed to provide better patient care. key words: Full-text PDF: Word count: Tables: Figures: References: relaxation response • nitric oxide • belief • limbic system • norepinephrine • morphine • dopamine http://www.medscimonit.com/fulltxt.php?IDMAN=9437 5731 — 3 145 PE This copy is for personal use only - distrib This copy is for personal use only - distribution prohibited. This copy is for personal use only - distribution prohibited. This copy is for personal use only - distribution prohibited. py is for personal use only - distribution prohibited. © Med Sci Monit, 2006; 12(9): HY21-31 PMID: 16940938 Author’s address: Dr. George B. Stefano, Neuroscience Research Institute, State University of New York College at Old Westbury, Old Westbury, NY 11568, U.S.A., e-mail: [email protected] Current Contents/Clinical Medicine • SCI Expanded • ISI Alerting System • Index Medicus/MEDLINE • EMBASE/Excerpta Medica • Chemical Abstracts • Index Copernicus HY21 Electronic PDF security powered by IndexCopernicus.com Med Sci Monit, 2006; 12(9): HY21-31 One may speculate that cognitive abilities arose or evolved because highly complex sensory-motor integration mechanisms were already in place (an organized brain) on which this “highly developed” coping strategy could be based. Moreover, cognitive coping uses this foundation as its operating platform. That is, cognition must be able to activate normal non-cognitive stress phenomena as well as deactivate them at the appropriate time, i.e, relaxation. Indeed, the subtleties of the integration would be hard to discern, as would the exact stimulus of a complex sensory experience. Yet, this would not take away from the existence of the connection/integration, it only further dramatizes its complexity. Thus, for example, we can predict that both cognitive and non-cognitive coping would be able to influence immune phenomena via neuro-immune, vascular-immune and neuro-vascular mechanisms. Based on the above, the mind depends on the underlying neural substrates to manifest itself. Thus, both consciously and unconsciously, it should be able to influence its underlying foundation. R SO O N N A LY L U It is reasonable to propose that our bodies contain naturally occurring antibiosenescent, health maintaining and antistress processes, involving immune, vascular and neural systems, that serve to maintain our life for a reasonable period of time, and that the vigor of these processes, in part, determine our mean life span. In most mammals, once these protective systems diminish in their capacity, their reproductive life has also ended. In man, however, in part aided by our integrative capacity, our life span is extended well beyond our reproductive years. It would not be surprising, therefore, to find critical neuronal processes linked to man’s cognitive ability that have the ability to promote health. These constitutive processes would manifest themselves above background activity during times of need, e.g., stress, when an increase in a health-related cognitive stimulus initiates this innate, non-cognitive protective neural process to become evident. We speculate that the ability to relax is a major physiological process, which emerges when “conditions” are appropriate. or regulate the many individual neural processes that potentially summate a decision-making process. That is, the brain represents only neural tissues organized into various neural patterns that can work together or separately. Without a unifying component being able to cope with a focus, the significance and uniqueness of this coping strategy would be lost. These individual processes (storing sensory information and motor responses in many brain compartments, along with the multitude of simultaneous integration processes) are extremely complex and varied. Moreover, a unified entity, a “mind”, would only be involved with experience-related phenomena (both exteroceptive and interoceptive) since this is the realm in which coping strategies are designed [4]. SE BACKGROUND WHAT IS RELAXATION? For more than 30 years, Herbert Benson and colleagues, building on the work of Swiss Nobel Prize laureate Dr. Walter R. Hess, have described a physiological response, termed the “relaxation response”, that they describe as being the opposite of the stress response [1]. It results in decreased metabolism, heart rate, blood pressure, and rate of breathing, as well as a decrease in brain activity [2]. Recently, we have added nitric oxide (NO) as a molecule involved in this learned response [3–5]. Clearly, this response embodies actions that one would associate with relaxation. We surmise the relaxation response is part of a larger physiological process, which can simply be referred to as relaxation. In this regard, we also surmise that the ability of a practitioner or person to elicit the relaxation response is strengthened by the trust or belief in the expected outcomes as well as the belief that a particular environment is safe or suitable to relax [6]. In fact, the strength of the fiduciary relationship between physician and patient appears to play a direct role in the effectiveness of medical treatment [4,6–10]. Thus, it would appear that there are larger physiological processes allowing for relaxation to occur. However, the exact mechanism or combination of cascading mechanisms involved until now has escaped detection. PE This copy is for personal use only - distrib This copy is for personal use only - distribution prohibited. This copy is for personal use only - distribution prohibited. This copy is for personal use only - distribution prohibited. py is for personal use only - distribution prohibited. Hypothesis WHAT IS THE MIND? Before going further we must define the term “mind.” For us, it was enticing to think that the chance alteration of genetic or neural pathways leading to cognitive processes also provided such endowed animals with an additional survival coping strategy [11] and that these cognitive coping abilities provided such organisms with a competitive edge for survival. The burgeoning of cognitive theory and therapy in the recent past is testimony to the insight that altering and improving cognitive coping mechanisms can help dissipate the emotional ravages attendant to the stress response. Interestingly, cognition may also contribute to stress when it cannot be turned off [12]. In order for cognitive ability to develop and succeed, however, there must first be a unifying consciousness to control Belief has an emotional component in that the brain motivation and reward circuitry will be reinforced with a positive emotional valence attached to the believed in person, idea or thing [4,7–9,12,13]. This emotionalized memory, replete with “somatic markers”, i.e., bodily sensations that accompany emotion and set the feeling tone, “feels right” to the person [14]. Clearly, emotion can be viewed as a process reinforcing a belief so that rationality cannot “weigh” the belief down into a lack of activity (see [11,15]). Indeed, belief in regard to a therapy, doctor, or personal religion may stimulate physiological processes, enhancing naturally occurring “healthy” processes by augmenting their level of performance [11,15]. Furthermore, belief, trust, pleasure and love via limbic as well as other central nervous system (CNS) and peripheral nervous system (PNS) processes (see [4,7–9,13,16] for details on the hard wiring of these processes) are involved in relaxation because they are critical factors in the conscious or unconscious decision making that takes place in determining whether it is safe or not safe to relax (Figure 1). Given this, can a person’s belief in an expected outcome actually affect the expected outcome? We believe the answer is yes, barring organic disease in the sensory, integrative or motor component of the processing pathway. Thus, cognitive and non-cognitive neural processes originating in the brain/mind may communicate through simple signaling pathways to intimately link the CNS and the diffuse immune system, including vascular components. While many of the body’s processes occur without cogni- HY22 Electronic PDF security powered by IndexCopernicus.com Stefano GB et al –Neural processes in relaxation Cholinergic triggerNicotine enhances morphine release Dopamine First and Always Conscious and Unconscious Limbic Involvement Sympathetic Response Flight or Fight Stress Response Norepinephrine Rapidly Inhibits Morphine If appropriate – depends Nitric oxide on safe environment-Activation tive input, the use of cognitive intervention or awareness as a coping strategy allows us to manipulate non-cognitive processes, implying that the “mind” can intervene and impose change in physiological systems. Indeed, this change can be both beneficial and pathological. Non-cognitive processes, therefore, may exist to promote health and cognition itself may stimulate these health-promoting mechanisms when properly activated or called upon. Clearly, this type of “hard” non-cognitive and “soft” cognitive linkage is instrumental in the concept of the mind-body phenomenon. We believe it is also quite important in understanding relaxation, since it also may represent a physical manifestation of this phenomenon. Admittedly, these processes are called on in many animals in a very unconscious way, indicating that they may be of primal origin, i.e., antibiosenescent. STRESS HY R SO O N N A LY L U Relaxation Figure 1. As described in the text, it is surmised that the adrenergic/sympathetic system is always on, maintaining an alert status, which provides high survival value. However, after cognitive and non cognitive limbic associated neural processes have determined it is safe to reduce the level of alertness, i.e., relax, relaxation can proceed. This latter process may involve a cholinergic trigger, switching to morphinergic and subsequent nitric oxide signaling. This signaling suppresses further catecholamine metabolism to norepinephrine while simultaneously liming its signaling capabilities, i.e., nitric oxide inhibits dopamine beta hydroxylase. Thus, relaxation appears to result from general inhibition of the active sympathetic tone. SE CNS PE This copy is for personal use only - distrib This copy is for personal use only - distribution prohibited. This copy is for personal use only - distribution prohibited. This copy is for personal use only - distribution prohibited. py is for personal use only - distribution prohibited. Med Sci Monit, 2006; 12(9): HY21-31 Stress describes a challenge that forces biological organisms to react in order to adapt (keep balanced) and stay “healthy”, i.e., survive [17]. Simply defined, stress represents an event or stimulus that alters the existing immediate organismic homeostasis or “allostasis” [18]. Through an extremely complicated allostatic process, all living organisms maintain their survival in the face of both externally and internally generated “stressors”. This apparent harmonization is constantly challenged often to the point of threat [12,17,19–24]. Thus, the stress responses (physiological processes that occur in the face of stress, e.g., fightor-flight response) can be viewed as being highly protective. The broad spectrum of stimuli capable of engaging the stress response is remarkable and reflects how well integrated our perceptions of the physical, psychological and social worlds are [25]. Biochemical (neurotransmitter, peptides, steroids), physiological (heart rate, blood pressure) and behavioral (anxiety, depression, tension) concomitants of stress may co-mediate a disease response [26]. Another important element of stressful stimulation may be the duration or time component of the noxious stimulus [20,27]. A brief physical or mental “assault” may allow an organism, through various detailed allostatic compensatory mechanisms, to “deal” with both an appraised or perceived stress. If the situation were to continue chronically, the organism might become vulnerable, susceptible to negative aspects of the stress response, such as in the case of prolonged immune down-regulation [28–32]. Moreover, our physiological and psychological stress response “systems” plainly function or were designed to function over the short term, i.e., fight or flight, not for prolonged periods of time. Given the signal molecule commonalties and similarities found in diverse organisms during the course of evolution, not to mention the common design of animal nervous systems regardless of phyla [24,33–38], it is not surprising to learn they also similarly exhibit stress responses [24]. The pathological effects of stress are those induced by longterm stress. This is what Hans Selye referred to as the “general adaptation syndrome”, when a particular stressor (highly emotional situation, physical abuse, etc.) remains for a long period of time [39]. Here, the persistent elevated stress response causes the system to function at its full capacity. This cannot continue for extended periods of time without metabolic detriment to the organism [17,22,23,40]. Thus, short-term stress processes are beneficial because we can overcome a particular obstacle. Long-term expression of these processes can be viewed as detrimental. Our physiologic systems are not designed for long-term stress, such as prolonged immune compromise. We may also bring upon ourselves a long-term stress resulting from our perception of the stressor itself. Perhaps, with the appearance of cognitive appraisal capabilities, human beings were, as a side effect, able to translate the short-term stress process into a longerterm stress process simply by thinking about it and moreover dwelling on it (such as contemplating a boss firing you for several months, cognitive “constipation”) [12]. Furthermore, as just noted, chronic stress can impact many physiological systems. In part, the reason for this may be that at the core of many disorders one may find a proinflammatory situation that manifests itself in diverse tissues, differ- HY23 Electronic PDF security powered by IndexCopernicus.com DA, Various cellular compartments Requires Excitation/Activation NE Norcocolarine Reticuline Salutaridine Thebaine Codeine Morphine Morphine 6 glucuronide R SO O N N A LY L U E Requires down regulation/calm, restore homeostasis Activation-Maintain a state of high alertness, energy Down regulate the activation, restore homeostasis ently masking the commonality [41]. In this regard, various integrative medical techniques may be efficacious because they too exhibit commonalities [7–9] not only in regard to shared signal molecules, i.e., opioid, and neural circuit, i.e., limbic, components but their ability to depend on trust and belief, inducing a state of relaxation. Moreover, it may be the ability to induce relaxation that breaks the negative impact of chronic stress [12]. Thus, it is not surprising to find that integrative/complementary medical techniques appear to have a general global impact on diverse disorders [42– 44] given the relationship they have with stress, i.e., calming or stress reduction. RELAXATION Figure 2. The “Ying-Yang” nature of the excitation and relaxation pathways manifests itself in the biochemical pathway for synthesizing dopamine and morphine, both arising from common precursors. Despite the common precursor one pathway initiates activation whereas the opiate pathway initiates down regulation and mediates pleasure and reward signaling that can only emerge once no life threatening phenomena are perceived. Given this common component of both pathways we surmise that endogenous auto regulators determine which activity emerges. This may also help explain the significance of emotion as a trigger for this pathway whose outcome depends on what emotion is evoked. It also, based on neuroanotomical data, provides a critical understanding for profound behavioral modifications that can occur in addiction, for example [7,8,13,42,43,112,117,118,143]. SE DOPA Tyramine PE This copy is for personal use only - distribution prohibited. This copy is for personal use only - distribution prohibited. py is for personal use only - distribution prohibited. Med Sci Monit, 2006; 12(9): HY21-31 Tyrosine This copy is for personal use only - distribution prohibited. This copy is for personal use only - distrib Hypothesis (cNOS) is stimulated, NO release occurs for a short period of time, but this level of NO can exert profound physiological actions for a longer period of time [32,45]. NO is not only an immune, vascular and neural signaling molecule, it is also antibacterial, antiviral, scavenges free radicals, and it down-regulates endothelial and immunocyte activation and adherence, thus performing vital physiological activities, including vasodilation [32,46–48]. Thus, NO has the potential to protect an organism from microbes and physiologic disorders such as hypertension, and also diminishes excessive immune and endothelial activation [3,46]. Indeed, its constitutive presence may set the tone for the activation of these cells and its absence or presence at lower levels may set the stage for progressive deterioration, i.e., Alzheimer’s Disease (see [3,45,49]). NO activity Briefly, we surmise that relaxation represents an equally real physiological process, allowing for physiological recovery and promoting health by terminating the other physiological process normally associated with stress (Figure 1). In this regard, it represents a naturally occurring proactive protective mechanism that, once stimulated, provides a beneficial outcome for the individual invoking the process [42–44]. We surmise that the molecular components of this process must be constitutively expressed so that it can continually be “felt” as well as be stimulated to rapidly increase its desired beneficial effect. This aspect of relaxation is important in terminating, for example, alert states such as found in the stress response once a perceived threat ends (Figure 1). The inability to invoke relaxation may be an important factor in allowing chronic stress to gain a foothold in a person’s mind translating into pathophysiological disorders [12]. Background molecular processes We surmise constitutive NO signaling is critical in relaxation [12,32,40]. When constitutive nitric oxide synthase The significance of cNOS-derived NO may also be ascertained by the number of chemical messengers that can stimulate its production. For example, the endocannabinoids, anandamide and 2-arachidonyl-glycerol (2-AG), are naturally occurring cNOS-derived NO-stimulating signaling molecules that are also constitutively expressed [32,50–61]. Anandamide, an endogenous endocannabinoid, can also cause NO release from human immune cells, neural tissues and human vascular endothelial cells [53,62]. Anandamide can also initiate invertebrate immune cell cNOS-derived NO [50]. Estrogen can also stimulate cNOS-derived NO in human immune and vascular cells [62–64]. It can also perform this function in invertebrates, stimulating cNOS-derived NO release from neural tissues [65,66]. Recent work from our laboratory has revealed that morphine can be made by normal healthy animal cells, including human cells [67,68] (Figure 2). This supports the previously found opiate receptor subtype μ3, cloned from human immune, vascular and neural tissues, which is selective for HY24 Electronic PDF security powered by IndexCopernicus.com Stefano GB et al –Neural processes in relaxation Morphine μ3 Blood Vessel Endothelium Nitric oxide WBC HY R SO O N N A LY L U Neuron Vasodilation Down regulate proinflammatory events Antibacterial, antiviral, scavenges free radicals Inhibits norepinephrine synthesis Inhibits immunocyte adherence Inhibits smooth muscle contraction Activates Reward Centers - Feels Good Calms Nervous tissues Figure 3. Peripheral manifestations of molecular components of relaxation. In order to be effective and have the potential to exert a therapeutic outcome relaxation must be broad in its ability to induce widespread down regulation while simultaneously not completely altering an animals excitatory alert processes to the point where they are rendered useless. Hence it not surprising to find the mu3 opiate receptor, which is opiate alkaloid selective and opioid peptide insensitive, present on diverse tissue types where it is coupled to constitutive nitric oxide release, which we also have linked to global down regulation and thus, exerting therapeutic actions that are equally diverse [42,43,43,118,119,144,145]. SE CNS and Adrenal Gland Acetylcholine the opiate alkaloids morphine and morphine-6-glucuronide and not opioid peptides [69], demonstrating a specific morphinergic signaling mechanism in animal tissues. Furthermore, μ3 opiate receptors stimulated by morphine are coupled to cNOS-derived NO release, resulting in the down-regulation of immune, vascular, gut and neural tissues [3,70–74]. Thus, besides well known cNOS-derived NO stimulators, i.e., acetylcholine (ACh) [75,76], newer chemical messengers have been discovered, which are also associated with NO coupling. Why are there so many pathways that lead to cNOS-derived NO release? We believe that each signaling system performs this common function under different circumstances. Endogenous morphine [32], given its long latency before increases in its levels are detected, arises after trauma/inflammation and, through a NO mechanism, downregulates these processes in neural, vascular and immune tissues [28,55,77]. Anandamide, as part of the ubiquitous arachidonate and eicosanoid signaling cascade, serves to maintain and augment tonal NO in vascular tissues [32,59]. Estrogen, through NO release, provides an additional pathway by which the system can down-regulate immunocyte and vascular function in women [62]. This may be due to both the immune and vascular trauma associated with cyclic reproductive activities, such as endometrial buildup, when a high degree of vascular and immune activities are occurring. Given the extent of proliferative growth capacity during peak estrogen levels in this cycle, NO may function to enhance down-regulation of the immune system to allow for these changes. Clearly, therefore, enhanced cNOS activity is beneficial within the concept and time framework of relaxation. PE This copy is for personal use only - distrib This copy is for personal use only - distribution prohibited. This copy is for personal use only - distribution prohibited. This copy is for personal use only - distribution prohibited. py is for personal use only - distribution prohibited. Med Sci Monit, 2006; 12(9): HY21-31 Proposed signaling in relaxation Individuals who are relaxing experience peripheral vasodilation, warming of the skin, a decrease in heart rate and an overwhelming sense of well-being only when this can occur in a safe and trusted environment (see [4]). Counter-intuitively, there may be initial sympathetic activation, as noted by norepinephrine (NE) levels, which initially go up [78]. This appears also to be the case for falling in love and enjoying pleasurable experiences, since this does, for example, represent the risk of rejection [8,13,79,80]. Regarding the vasodilator peripheral heat-warming processes, we speculate that this involves NO [4]. In regard to the sense of well-being, we can assume that this process may also involve opioid signaling molecules, which are involved with reward processes [8]. In examining a potential mechanism for relaxation, besides the over-riding CNS output via the autonomic nervous system, peripheral neuro-vascular processes would appear to be important [4] (Figure 3). We surmise NO to be of fundamental importance because of the increase in peripheral temperature, i.e., vasodilation [81]. With reference to the peripheral vasculature, we find nerve terminals in the vessels that when stimulated by nicotine, result in vasoconstriction followed by vasodilation [82], indicating a cholinergic mechanism. Clearly, this phenomenon is in line with new data demonstrating nicotine stimulation of endogenous morphine from nervous tissue, resulting in morphine NO coupling and vasodilation, i.e., relaxation [78,83–86]. The vasoconstriction component of the biphasic nicotine effect is mediated by alpha-1-adrenoceptors stimulated by NE liberated from peripheral sympathetic adrenergic nerves [4]. Studies suggest that, because of insensitivity to atropine, ACh does not mediate the nicotine-induced vasodilation [82]. Instead it is mediated from nerve endings in which a NO generating system and ACh may coexist [87]. Thus, nicotine stimulates the adrenergic and nitroxidergic nerves innervating, for example, the temporal arterial wall of denuded endothelium in superficial dog tissue, resulting in contraction and a rapidly developing vascular relaxation, the latter being mediated by cyclic GMP (cGMP) [88]. Slow relaxation caused by nicotine is associated with the elevation of cGMP production via activation of guanylate cyclase, which appears to be mediated by prostaglandin I2 [88]. However, newer experimental results allow us to modify this interpretation. Namely, nicotine can stimulate the release of HY25 Electronic PDF security powered by IndexCopernicus.com Med Sci Monit, 2006; 12(9): HY21-31 ly, the relaxation response [78] may, in a sense, represent the left over flow of the earlier mild sympathetic stimulation, i.e., also representing work initiation in a different situation, i.e., stress response. SE Complicating this matter is the data indicating that ACh inhibits, acting via prejunctional muscarinic, not nicotinic receptors, the synthesis and release of NO while concurrently antagonizing the release of NE [92,95]. However, the response to exogenous NO is not influenced by ACh [93]. In addition, the secondary vasodilation to electrical nerve stimulation appears to be attenuated by treatment with ACh in a concentration-dependent manner [93]. These findings suggest that ACh plays an essential role in vascular NO regulation (Figure 3). It may be the factor adjusting the interaction of NO and NE, simultaneously exerting its own vascular action, i.e., stimulating endothelial NO release maybe via the release of endogenous morphine [83,89,106,107] (Figure 3). Others found that beta(2)-adrenoceptor antagonists blocked the relaxation induced by nicotine. Furthermore, they demonstrated that beta(2)-adrenoceptor immunoreactivities and NADPH diaphorase reactivities were colocalized in the same nerve fibers in basilar and middle cerebral arteries [103]. The authors speculate that NE acts on presynaptic beta(2)-adrenoceptors located on the NO nerve terminals to release NO resulting in vasodilation. R SO O N N A LY L U endogenous morphine from adrenal tissues and white blood cells, adding this opiate alkaloid into the immediate environment [83,89,90]. It is critical to note that, as documented earlier, endogenous morphine signaling is coupled to cNOSderived NO release in these tissues. In individuals shown to relax via lowering their blood pressure by listening to music, higher opiate alkaloid levels were found in their plasma along with a significant alteration of the μ3 receptor levels on blood cells and a lowering of plasma proinflammatory cytokine levels (i.e., immune down-regulation) [86,91], demonstrating the involvement of opiate alkaloids in this ability to relax at the peripheral level. Supporting this hypothesis further is the vasodilator effect, which requires NO, resulting in the lowering of blood pressure and the ability of morphine to release NO from immune and vascular cells as discussed earlier. This release may be the actual event contributing to the observed vasodilation and increase in peripheral skin temperature associated with relaxing (Figure 3). Taken together, we surmise that NE initially promotes a slight vasoconstriction of the artery during the initial phase of relaxation (“getting in the mood”), indicating a slight enhancement of sympathetic activity upon stimulation (Figure 1). This may also serve to demonstrate that sympathetic tone always manifests itself until its influence is diminished. This is immediately followed by the release of NO from the nitroxidergic nerve or from an immune and/or vascular source, which mediates a concentration-dependent vasodilation. In monkeys, the cerebral arterial diameter under resting conditions is maintained by tonic release of NO from the nerve (10–20%) or from the nerve and endothelium (30%) [92]. This observation is supported by other data from our laboratory since basal NO is cNOS-derived and keeps particular types of cells in a state of inhibition [3]. Endogenous superoxide dismutase (SOD) in the cerebral artery appears to protect the relaxation, induced by NO from perivascular nerves, from the NO scavenger action of superoxide anion [93]. We surmise that this NO then produces the longer-lived phenomenon of smooth muscle relaxation. In another report, it was found that NE vascular hyper-responsiveness in hypertension is dependent on an impairment of NO activity that is realized through NE-induced oxygen free radical production [94], demonstrating an important contribution to the understanding of this regulatory process. PE This copy is for personal use only - distrib This copy is for personal use only - distribution prohibited. This copy is for personal use only - distribution prohibited. This copy is for personal use only - distribution prohibited. py is for personal use only - distribution prohibited. Hypothesis In regard to a nervous origin of relaxation, the location of the NO-releasing nerve, nitroxidergic nerve, has continued to be a subject of debate. Various findings lead us to believe that the nitroxidergic nerve is located in the proximity of the adrenergic sympathetic nerve bundle [88,95–101]. Furthermore, the NE-stimulated vasoconstriction is followed by relaxation that is suppressed by L-NA, a NOS inhibitor [102], supporting its interaction via a NE mechanism [103]. Secondly, nicotine-induced relaxation is abolished by guanethidine, tetrodotoxin, and pretreatment with 6hydroxydopamine, all causing destruction of the sympathetic nerve, and demonstrating again the NE component [104]. Furthermore, we have demonstrated that eNOS-derived NO can inhibit NE neural release in animal vasculature [53]. Recently, we have also demonstrated that, once NO is present, smooth muscle cells from rat and human arteries fail to contract in response to NE [105], demonstrating that when the balance shifts to NO, NE cannot initiate vasoconstriction. Thus, the NE reported in, specifical- We also surmise, based on current studies, that endothelialderived NO, released through normal pulsations due to vascular dynamics responding to the heart beat [32,73,108], as well as ACh-stimulated endothelial NO release (via nicotinic processes), may contribute to the effect of NO in inhibiting NE processes as well as inducing smooth muscle relaxation. Furthermore, vascular pulsations may be of sufficient strength to also stimulate neuronal NOS-derived NO release, limiting any basal NE actions. Interestingly, nitrosative stress, mediated by involvement of the reactive nitrogen oxide species N2O3, does inhibit dopamine-b-hydroxylase, inhibiting NE synthesis and contributing to the regulation of neurotransmission and vasodilatation [109,110]. This system may provide an autoregulatory mechanism involved in the neuronal control of peripheral vasomotor responses. Furthermore, the ability of nicotine to release morphine from adrenal and immune tissues helps explain the source of NO [83,89,90], which results from the newly released morphine and μ3 coupling to NO release on these critical tissues. In conclusion, relaxation peripherally appears to be mediated by a system of regulation involving NO, NE, ACh and morphine as neurotransmitters and local hormones. Contingent on the preliminary vasoconstriction and depolarization of the membrane initiated by the release of NE, vasodilation is mediated by NO liberated from vasodilator nerves and maybe other tissues that activate guanylate cyclase in smooth muscle and produce cGMP. During this stage, NO and NE exist simultaneously. Due to the signaling cascade of NO, NE no longer mediates vasoconstriction. Instead, NO activates guanylate cyclase, which produces vasodilation and the relaxation under a depolarized membrane state. CNS involvement The CNS regulating pathways and processes integral to mediating this process stimulate NE release either alone or in HY26 Electronic PDF security powered by IndexCopernicus.com ated release of NO from rat brain hippocampus and amygdala and endogenous morphine’s presence in these tissues [117]. Furthermore, DA can serve as a endogenous morphine precursor [67,68] (Figure 2). HY Supporting this hypothesis are other studies linking relaxation characteristics with that of pain. Once individuals are exposed to painful stimuli such as a penetrating needle they experience peripheral vasodilation, warming of the skin [123–126], an increase in heart rate and a sense of agitation, all as a potential result of circulating catecholamines and/or NO [127]. It is the function of the amygdala and related subcortical regions to aid in the relief of these pain states [113,128,129]. In examining a potential mechanism for this relief, besides the over-riding CNS output via the autonomic nervous system, peripheral neuro-vascular processes would appear to be important. Again, we surmise that NO and its relation with prostaglandins are of fundamental importance in this response because of the increase in peripheral temperature, i.e., vasodilation, in the ensuing inflammatory response [123–126]. Indeed this is precisely the pathway, which is inhibited with non-steroidal antiinflammatory drugs (NSAID). Interestingly, as noted earlier, N2O3 does inhibit dopamine hydroxylase, inhibiting NE synthesis and contributing to the regulation of neurotransmission and vasodilation [4,110]. This system may provide an autoregulatory mechanism involved in the neuronal control of peripheral vasomotor responses as they relate to pain control, where induction of a catecholamine-inhibiting substance may autoregulate the vasomotor response in response to nitrosative stress (see [130] for the role of nitrosative stress in neuropathic pain). R SO O N N A LY L U There are several key areas of the CNS involved in relaxation processing. Subsequent to thalamic routing, there is quick transit of crude data to the amygdala and a slower more considerate flow of information to the primary sensory cortex, with subsequent routing of more refined information to the amygdala and the hippocampus, areas classically involved in emotion and memory – critical functions of the so-called limbic system of the brain (Figure 1). The quick route allows the organism to be rapidly responsive to a fearful prompt. The more sophisticated appraisal route permits the limbic system to operate with more reliable information that may allow the amygdala to stand down and for relaxation to re-emerge if the decisions to be made are safe and clear cut ones. If the decisions to be made are not clear cut and are conflicted in some way the anterior cingulate cortex must make a response selection based on the cognitive data from cortical regions and the emotional data from the limbic system [111]. Hyperactivation of the anterior cingulate as seen in functional neuroimaging of depression and in pain states can be modified when the prefrontal cortex is stimulated by conscious positive expectation and the relaxation that accompanies it [112]. Stefano GB et al –Neural processes in relaxation SE combination with NO (see [4]). If NE is released alone, vasoconstriction occurs. If NE is released with NO, we surmise an initial short-lived vasoconstriction occurs followed by a prolonged vasodilation mediated directly by NO. In this regard, if basal/tonal NE is present, NO overrides this effect (Figure 1). If ACh is present, we surmise that its inhibition of NE, causing loss of sympathetic tone, and its stimulation of endothelial-derived NO, may also result in vasodilation. The important point at this stage of explaining relaxation is that, at last, we are beginning to see a mechanism that can explain its characteristics and simultaneously provide an explanation for its health benefits, i.e., via cNOS-derived NO (see [3,32,45,47,48]). Hence, it is no surprise that these limbic and paralimbic areas are of great importance to relaxation, as well as pain modulation. We surmise the proximity and shared “wiring” of these two functions represents a critical point whereby evaluations are made on whether or not the situation is appropriate to relax. This proximity and sharing of common neural circuits is a critical survival element in animal evolution, allowing excitation to take over rapidly should the situation arise. Importantly, the central nucleus of the amygdala is most strongly modulated by dopamine (DA), NE, epinephrine and serotonin [113,114]. The basal nuclei receive moderately high inputs of DA, NE and serotonin [113,114], each of which has been demonstrated to exert their desired effect, in part, via NO [115–119]. PE This copy is for personal use only - distrib This copy is for personal use only - distribution prohibited. This copy is for personal use only - distribution prohibited. This copy is for personal use only - distribution prohibited. py is for personal use only - distribution prohibited. Med Sci Monit, 2006; 12(9): HY21-31 We surmise that the centrally released NE exerts effects in the periphery via cardiovascular circuitry [4] by initially promoting a slight vasoconstriction of the peripheral artery during the amygdalar response to relaxation input as part of the limbic system’s inherent mechanism to maintain homeostasis and decrease pain perception via descending spinal pathways resulting in endogenous opiate analgesia [115,116]. In fact, reports have found endogenous morphine within the structure of the hippocampus and amygdala, i.e., limbic system [117,120–122]. In addition, this morphine may activate pleasure pathways via NO in rat brain hippocampus [112]. Studies from our laboratory confirm the medi- This line of research allows us to further speculate that physiological relaxation emerged from this pathway, once the proinflammatory element, under the right circumstances, was removed. This probably was associated first with physiological states requiring a less alert state, such as sleep-like activity. Later, when cognition evolved, it also became associated with our ability to determine a safe environment in which relaxation may emerge. In either event, it would be critical to have endogenous morphine involved because of its analgesic action and ability to down-regulate immune processes [30,32], effectively separating the two functions, which would be critical to relaxation. Indeed, cognitive functions and devices that show their performance (EEG, fMRI, SPECT, etc.) reveal a clear distinction between sleep and relaxation, and relaxation not only leads to an overall decrease in brain activity but simultaneously to an increase in specific parts of the brain, e.g., related to cognition [131–133]. Another critical component of this determination, whether to be alert or relax, is having morphine synthesis take place via the catecholamine pathway in animals, including man [67,68]. Again, this pathway behaviorally allows for excitation to occur as synthesis proceeds via DA and secondarily NE, followed by down-regulation via morphine and morphine-6-glucuronide signaling, as it goes to its end at opiate alkaloid synthesis [67,68]. THE HYPOTHESIS cNOS-derived NO appears to be critical for relaxation because it has the ability to block a sympathetic response simply by having its release occur beyond the basal level, which HY27 Electronic PDF security powered by IndexCopernicus.com Med Sci Monit, 2006; 12(9): HY21-31 In speculating on an overall theory as to what constitutes relaxation, we offer the following hypothesis: Neural processes (CNS integrating limbic circuits), involving NO and morphine, activate downstream signaling molecules that stimulate cNOS-derived NO release from immune, neural and vascular tissues. Prior to NO release this process also invokes the release of NE and opioid peptides. The presence of NO is deduced by its vasodilating actions and the lack of vascular sensitivity toward NE and by direct measurement [5]. Relaxation can be cognitively learned, e.g., relaxation response. Taken together, this potential is always present and only after removing the sympathetic and stress reactions (e.g., disinhibition [3]), does it emerge. It is probably this process along with many others that provide for mammalian longevity, i.e., it is antibiosenescent. R SO O N N A LY L U Additionally, endogenous morphine signaling appears to play a major role in relaxation. Many immune processes perpetuate and become embellished with time by the recruitment of cells, and through beneficial yet sometimes harmful signaling molecules such as the proinflammatory cytokines. These molecules can all be down-regulated by morphine, which is released following stress or trauma [30,32], specifically through cNOS-derived NO under certain circumstances. Thus, morphine may help overcome over-stimulated immune, vascular and neural tissues [32,68]. As such, it may be part of the immune system regulation that prevents the all too common ravages of what Bone called “immunologic dissonance”, including the systemic inflammatory response syndrome (SIRS) which sometimes culminates in the often lethal multiple organ dysfunction syndrome (MODS) [134]. burst of NO, we can enhance the inhibition of NF-kB activation, limiting the extent and the severity of a proinflammatory situation. Does this occur during long-term stress or does the proinflammatory mechanism become desensitized and over-stimulated by a relentless insult, so that NO is formed through inducible NOS, which not only becomes detrimental to the cells, but to the entire organism? We believe the latter situation emerges with chronic stress and may give rise to certain degenerative diseases [17,22,23,40,139]. For example, if a proinflammatory challenge is overwhelming or persistent, iNOS production may reflect a last ditch attempt to overcome antigenic stress while also trying to dampen a potentially destructive proinflammatory blaze. The resulting overproduction of NO is unfortunately toxic [4]. SE will lead to vasodilation and peripheral sense of warmth. Its presence can also explain the paradox of the presence of NE in plasma while vasodilation is taking place [78]. Recently, we demonstrated that during the relaxation response NO levels are increased [5], further supporting the critical role of NO in this process. It is possible that some individuals may be deficient in this regulatory process, leading, when challenged, to the unregulated and potentially damaging immune responses of SIRS and MODS. We have found, for example, that in the immune disorder histiocytic medullary reticulosis or malignant histiocytosis [135] a morphine-selective receptor, μ3, was not expressed, and granulocytes and monocytes cultured from this patient could not be down-regulated when exposed to morphine. The foundation of the concept that morphine is critical to relaxation or a down-regulation of excitatory processes is supported by these preliminary clinical findings, as well as the fact that it can be made by human white blood cells and the μ3 opiate receptor subtype is found on these tissues as well [68,69]. Furthermore, high morphine levels have been found in rat limbic tissues associated with NO release [117]. ASSOCIATED MOLECULAR MECHANISMS PE This copy is for personal use only - distrib This copy is for personal use only - distribution prohibited. This copy is for personal use only - distribution prohibited. This copy is for personal use only - distribution prohibited. py is for personal use only - distribution prohibited. Hypothesis The physiological significance of cNOS-derived NO, and for that matter morphine-stimulating NO release, is that it can influence proinflammatory and stress situations, presumably bringing both the “acute phase response” and the “acute stress response” under control [45,54,55]. We propose that cNOS-derived NO initiates these events in part by its ability to modify the function of the transcription factors, i.e., NF-kB [136–138]. NF-kB binding sites are present in the promoter regions of proinflammatory genes such as tumor necrosis factor (TNF), interleukin (IL)-1 and IL-6 (see [136]). Interestingly, stimulation of cells with proinflammatory cytokines leads to the degradation of the NF-kB inhibitor IkBa, liberating NF-kB. NF-kB is then free to activate the transcription of some of these very same proinflammatory cytokines. NO inhibits the expression of proinflammatory cytokines by stabilizing the NF-kB – IkBa complex, therefore preventing translocation of NF-kB into the nucleus [136,138]. NO can also interfere with the binding of NF-kB to the promoter region of proinflammatory cytokines such as IL-6, which causes T-cell proliferation [136,138]. Putting this in perspective, if we liberate molecules that have the ability to stimulate constitutive NOS, producing a quick Its expression probably also differs among individuals, and the strength of one’s beliefs or an animal’s individual history may also exert a profound impact on the heightened expression of these proactive innate protective responses that originate from the CNS. Relaxation takes place in that emotionalized transitional relationship found in a person when we are in a trusted and safe environment. Here, belief is critical. Once this condition is met then, and only then, will relaxation occur. 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