Spinal modulation of the muscle pressor reflex by nitric oxide and acetylcholine
Introduction
Muscular work, e.g., exercise, can elicit profound increases in cardiovascular function. These cardiovascular changes are mediated by increases in sympathetic activity and decreases in parasympathetic activity 25, 43, 44, 45, 64. Two neural mechanisms are thought to play an important role in causing the autonomic changes: (1) a reflex arising from the contracting muscle, and (2) descending input from higher brain centers. The basic tenet of the first mechanism, also called the “exercise pressor reflex” or the “muscle pressor reflex” (MPR), is that muscle afferent fibers are activated by the muscular contraction, and these neurons in turn reflexly stimulate the sympathetic neurons innervating the heart and vasculature 25, 43, 44, 45, 64. The basic premise of the second mechanism is that the sympathetic nervous system is activated in conjunction with the motor system, the signal or signals originating in the brain 43, 56. The second mechanism is termed “central command.” These two neural mechanisms are not mutually exclusive and both are thought to be involved in mediating the cardiovascular changes seen during muscular work.
This review will focus on the reflex neural mechanism as it pertains to static muscular contractions, i.e., the MPR. For the studies described in this review, a sustained isometric (static) contraction is evoked in anesthetized cats. In this model, the triceps surae muscle of one hindlimb is contracted by electrically stimulating the efferent motor neurons innervating the muscle. In turn, this contraction evokes reflex increases in cardiovascular function, i.e., heart rate (HR) and mean arterial pressure (MAP) rise. It has been shown repeatedly that the rise in HR and MAP evoked by the static contraction is abolished when the afferent input is interrupted 11, 34, 35, 37, 62, 67, indicating that indeed the cardiovascular changes are a reflex arising from the contracting muscle. The muscle afferent neurons responsible for the MPR are the thinly myelinated group III and the unmyelinated group IV fibers 25, 27, 37. These afferent neurons are responsive to the mechanical and metabolic changes that occur in the contracting muscle 27, 29, 30, 42. The mechanoreceptors and metaboreceptors that initiate the MPR synapse in the dorsal horn of the spinal cord 25, 58, 64. The mechanoreceptor component can be more precisely investigated by passively stretching the triceps surae muscle. This procedure, muscle stretch, activates muscle mechanoreceptors and evokes reflex increases in cardiovascular function 27, 30, 34, 35, 36, 52, 69. Thus, the dorsal horn serves as the first synapse for the MPR regardless of whether it is elicited by static contraction or passive stretch of the triceps surae muscle.
Over the past several years, we, and others, have been interested in the dorsal horn mechanisms responsible for mediating and/or modulating the pressor reflex. Figure 1 depicts a working model of the dorsal horn mediators that are thought to be important for activating the second-order neurons involved in the MPR. Static contraction of the triceps surae muscle increases the dorsal horn concentration of the excitatory amino acids (EAA) glutamate (GLU) and aspartate (ASP) [16]. In addition, the concentration of the neuropeptide substance P (SP) is also elevated 3, 39, 46, 62, 63. These increases are abolished if the afferent input is eliminated and/or if the contraction is prevented by paralyzing skeletal muscle 16, 62, 63. The EAA appear to excite dorsal horn cells via the n-methyl-D-aspartic acid (NMDA) and non-NMDA receptors. SP appears to act on neurokinin-1 (NK-1) receptors. Support for this arises from studies showing that blockade of NMDA, non-NMDA or NK-1 receptors in the dorsal horn attenuates the MPR 2, 17, 18, 23, 24, 26, 28, 59, 65, 68. This should not be interpreted as a final or complete picture though. It is possible that other neurochemicals and/or receptors are very important in mediating the MPR at the level of the dorsal horn. This is particularly germane because the number of synapses involved in the dorsal horn processing of the MPR is not known. Nevertheless, there is considerable evidence for the aforementioned neurochemicals and receptors, thus the basis for the current model depicted in Fig. 1.
However, Fig. 1 fails to depict the complexity of the processing because it is simply a two-dimensional representation of a single synapse. Afferent fibers typically branch upon entering the spinal cord, sending projections rostrally and caudally 10, 12, 47, 57. In turn, these branches send collateral branches that synapse on dorsal horn cells at more than one segmental level 10, 12, 47, 57. We have demonstrated that multiple spinal segments are involved in the spinal processing of the sensory arm of the MPR, even when the afferent input is limited to one segment 17, 18, 59, 65. NMDA, non-NMDA, and NK-1 receptors are involved in this, as depicted in Fig. 1. Thus, the dorsal horn is an important integrative site for the MPR and multiple spinal segments are involved.
Numerous neurochemicals and their respective receptors are involved in modulating the MPR at the level of the dorsal horn. These include (see Fig. 1) serotonin (5-HT), α2- adrenergic receptors, opiates, vasopressin, and oxytocin 3, 20, 21, 22, 39, 46, 53, 54. Activation of the receptors depicted in Fig. 1 reduces the magnitude of the MPR, indicating that enhancing cardiovascular function via muscle afferents can be modulated, at least pharmacologically, by activating receptors that are also known to modulate other types of sensory processing, e.g., pain. Thus far, there is only evidence that opiate receptor activation works, at least in part, by pre-synaptically reducing neurochemical release [39]. It is interesting to note that inhibiting vasopressin and oxytocin receptors enhances the MPR 53, 54. This suggests that these neuropeptides are tonically released and thus exert a tonic inhibitory action on muscle afferent processing in the dorsal horn. Alternatively, the muscular contraction may activate neurons that release these compounds, thus buffering the magnitude of the cardiovascular changes. Which of these mechanisms predominates is unknown at present. As described below, we have recent experimental data to indicate that acetylcholine (ACH) has a similar action. The fact that several different receptors found in the dorsal horn can, when stimulated, diminish the MPR underscores the important integrative role of this central nervous system (CNS) region as it pertains to the MPR.
For the nitric oxide (NO) and ACH experiments described below, microdialysis was used to deliver agents to the dorsal horn region. Microdialysis works on the principle of diffusion, and thus drugs are delivered to the tissue by perfusing the microdialysis probe with a solution containing a higher concentration of drug than is found in the tissue. Because the drug moves via diffusion, the tissue concentration and area of distribution are time and concentration dependent. For this reason, we typically dialyze drugs for at least 2 h. The microdialysis probe (BAS, CMA-10, West Lafayette, IN, USA) has a diameter of 500 μM at the membrane, which is 3 mm in length. The microdialysis probe or probes are inserted vertically into the dorsal surface of the exposed spinal cord using a Kopf carrier, and they are submerged such that the entire membrane is in the spinal tissue (the tip residing at ∼laminae VII). Based upon dye distribution or Prussian blue reactions, the entire dorsal horn (laminae I–VI) is covered and the rostrocaudal distribution is ∼5 mm 3, 39, 46, 62. In the NO study, two probes were inserted into the spinal cord, one at L6, and one at S1. For the ACH study, a single probe was inserted at the midpoint of the rostrocaudal extent of L7. Because sympathetic preganglionic neurons terminate at ∼L3, direct alterations in sympathetic transmission in the spinal cord is not an experimental complication in this preparation.
As indicated above, activation of dorsal horn NMDA receptors is involved in producing the MPR. The NMDA receptor is unique in that it is both ligand and voltage gated 5, 74. Binding of an EAA to the NMDA receptor opens a Ca2+ channel, which in turn evokes an excitatory post-synaptic potential 5, 48, 74. Calcium entry into the cell can activate a variety of cellular events, one of which is the activation of nitric oxide synthase (NOS), the enzyme responsible for NO synthesis. Recent evidence indicates that activation of NMDA receptors produces NO in neural tissue 13, 31, 38, 40, 50, and NOS is located in the superficial dorsal horn 8, 9, 55. There is considerable evidence that both NMDA receptors and NO are involved in the sensitization of dorsal horns that occurs with peripheral inflammation 32, 33, 38, 40, 41, 51, 70. Thus, activating NMDA receptors may liberate NO, which in turn increases the excitability of dorsal horn cells to peripheral input.
Because NMDA receptors are involved in producing the MPR and have been linked to inducing NO production, we hypothesized that NO is also involved in mediating the MPR. To test this, the NOS inhibitor L-NAME was delivered to the dorsal horn ipsilateral to the contracting muscle using the technique of microdialysis [61]. Figure 2 shows the time course of the rise in MAP elicited by static contraction before and during the dialysis of L-NAME into the dorsal horn. Prior to L-NAME, static contraction increased MAP. However, after 2 h of L-NAME dialysis, the curve depicting the initial component of the rise in MAP was suppressed. The maximal change in MAP was unaffected, but the initial change in MAP was diminished. In other words, static muscle contraction increased MAP the same amount before and after L-NAME, it just took longer to get to that point when NO production was inhibited. This is more dramatically illustrated in Fig. 3, which shows the magnitude of the change in MAP at 10 s before and during L-NAME administration. Note that L-NAME decreased the change in MAP at 10 s at both the 1-and 2-h time points. Neither the time course nor the change at 10 s was altered when an equimolar concentration of the inactive isomer of L-NAME, D-NAME, was dialyzed into the dorsal horn horn [61]. Thus, inhibiting NO production has a subtle effect on the MPR in that it diminishes the rate of rise in MAP that occurs in response to static contraction of skeletal muscle. These data suggest that abolishing dorsal horn NO reduces the sensitivity, at least to the initial input, of dorsal horn cells receiving input from muscle afferent neurons that are activated during a contraction.
Using a separate group of cats, we performed essentially the converse experiment: the substrate for NO, L-arginine, was dialyzed into the dorsal horn at the same spinal segments and at the same dose [61]. An original record from one cat is depicted in Fig. 4. This tracing shows that the increases in HR and MAP produced by the static muscle contraction are enhanced after ∼1 h of L-arginine dialysis. The mean data for the time course of the MAP changes before and during L-arginine is depicted in Fig. 5. Note that after 1 and 2 h of L-arginine administration, the MAP response is enhanced. L-arginine also enhanced the HR changes evoked by static muscle contraction. (It should be noted that all of the drugs used in this study were dissolved in an artificial buffer solution that has an ionic content similar to the extracellular space and the pH was adjusted to 7.4). Thus, administering L-arginine, and thus presumably increasing NO production, enhanced the MPR [61]. These data support the hypothesis that NO increases the excitability of dorsal horns cells involved in the reflex control of cardiovascular function.
Additional support for this concept can be found in experiments in which the MPR was evoked by stretching the triceps surae muscle. L-NAME had no effect on the stretch-induced MPR, while L-arginine enhanced the MPR [60]. Thus, increasing the substrate for NO appears to enhance the excitability of dorsal horn cells that are coupled to autonomic output and muscle afferent input.
The dorsal horn of the spinal cord contains cell bodies that stain positive for choline acetyltransferase, thus indicating that intrinsic spinal neurons synthesize the neurotransmitter ACH 4, 49. These cell bodies are typically found in the deep layers (laminae V–VI) of the dorsal horn, but axons and dendrites within the superficial dorsal horn also stain positive for choline acetyltransferase 4, 49. In addition, muscarinic receptors are found in the dorsal horn of the spinal cord 14, 15. At least some of these receptors appear to exist in presynaptic terminals because dorsal rhizotomy decreases their number [14]. On the other hand, transection of the spinal cord has minimal effects on the number of dorsal horn muscarinic receptors [15]. Considered together, this evidence indicates that the dorsal horn of the spinal cord contains a network of cholinergic neurons that may impact sensory processing in this region.
Physiological and pharmacological experiments suggest that activation of cholinergic receptors in the dorsal horn reduces sensory transmission in the dorsal horn 1, 7, 71, 72, 73. Intrathecal administration of atropine decreases the mechanical threshold for tail withdrawal without affecting the thermal threshold [73]. This suggests that ACH has a tonic inhibitory input with respect to the dorsal horn processing of input from high-threshold mechanoreceptors. Interestingly, it has been shown that activation of muscarinic receptors increases NO production in cultured sensory neurons from rats [6]. Further, intrathecal administration of L-NAME enhanced the atropine-induced reduction in the mechanical threshold for tail withdrawal, but NOS blockade had no effect when administered alone [73]. These data suggest that the muscarinic-induced reduction in sensory processing is dependent on NO production. This seems contradictory to the concept mentioned above, which is that NO enhances the excitability of dorsal horn neurons to peripheral input and thus enhances sensory transmission. Nevertheless, activation of muscarinic receptors in the dorsal horn appears to reduce sensory transmission.
We tested the hypothesis that increasing ACH levels in the dorsal horn reduces the MPR [19]. Figure 6 shows the effect of dialyzing the acetylcholinesterase inhibitor neostigmine on the peak increases in MAP and HR evoked by static muscle contraction and muscle stretch. Blocking acetycholinesterase increases ACH in the dorsal horn. This attenuation by neostigmine was abolished by atropine, suggesting that ACH blunted the MPR via activation of muscarinic receptors [19]. Not only did blocking muscarinic receptors eliminate the actions of neostigmine, but atropine treatment enhanced the MPR evoked by static contraction. An original record from one cat demonstrating this potentiation is depicted in Fig. 7. However, atropine failed to accentuate the MPR evoked by muscle stretch [19]. These results support the hypothesis that ACH reduces sensory transmission in the dorsal horn of the spinal cord, and it occurs in the processing of information that influences the cardiovascular system. Further, these data suggest that ACH, similar to vasopressin and oxytocin, has a tonic inhibitory action on dorsal horn cells receiving input from neurons activated by muscle contraction. Alternatively, afferent neurons activated by muscle contraction may excite cells, either directly or indirectly, that release ACH and these neurons in turn provide a feedback inhibition to the cells that are directly excited by muscle afferent fibers. At this point, it appears that this these latter two possibilities apply only to contraction-induced sensory transmission, since atropine failed to enhance the MPR induced by muscle stretch. However, caution is warranted since the atropine was only administered to the dorsal horn of only one spinal segment. Previous work has shown that blocking NK-1 receptors at only one spinal segment attenuates the contraction-induced increase in cardiovascular function, but has minimal to no affect on the stretch-induced response 65, 66, 68. However, when NK-1 receptors are blocked at two spinal segments, the MPR evoked response to stretch is attenuated [65]. Thus, more experiments are needed before firm conclusions can be drawn with respect to tonic or activation-induced muscarinic receptor modulation of stretch-induced cardiovascular changes.
Section snippets
Summary and future directions
The results from recent work provide additional support that the dorsal horn serves an important integrative function for the CNS processing involved in the coupling of skeletal muscle to the sympathetic branch of the autonomic nervous system. This is summarized in Fig. 1. The work involving NO provides preliminary evidence that the excitability of the transduction process for the afferent signal from skeletal muscle can be altered by cellular signals. In the working model depicted in Fig. 1,
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