Before the development of specific, antibody-mediated techniques, growth hormone (GH) activity in biological samples was measured by bioassay. One standard bioassay for GH measures growth of the tibial epiphysis in young, hypophysectomized rats (7). The advent of radioimmunoassay techniques in the 1950“s and recombinant DNA technologies during the1980“s facilitated considerable advancements toward understanding the physiological regulation of GH secretion and its roles in metabolism and somatic growth. During the 1970“s, Ellis and Grindeland (4) reported the existence of a potent pituitary growth factor that could not be measured by standard GH immunoassay but had GH-like activity in the tibial bioassay equivalent to 200–300 times the normal concentrations of circulating GH measured by immunoassay. They referred to this growth factor as bioassayable growth hormone (BGH) to distinguish it from immunoassayable growth hormone (IGH). Particularly compelling was the evidence indicating that the mechanisms regulating the secretion of these two pituitary growth factors could be disassociated. For example, differential modulation of BGH and IGH secretion occurred in response to physiological challenges such as fasting or cold exposure (4). Hymer, Grindeland, and colleagues (9) later reported further dichotomies between pituitary IGH and BGH secretion that were dependent on the gravitational environment. Following spaceflight, pituitary somatotrophs consistently secreted ∼50% less BGH in vivo and in vitro under both basal and GH releasing-factor–stimulated conditions, whereas no consistent changes occurred for IGH release. These results, in part, led to the hypothesis that proprioceptive alterations within the neuromuscular system might account for the changes in BGH secretion resulting from exposure to microgravity.
Recently, our laboratories have pursued this hypothesis and have demonstrated that a number of neurally-mediated stimuli can modulate BGH concentrations in rat plasma and pituitary tissue, as well as in human plasma. The purpose of this review is to summarize the evidence for a muscle afferent–pituitary axis. We propose that muscle afferents, perhaps specific to muscle spindles, can modulate BGH secretion from the pituitary. Such an axis would represent a novel component of the integrated neuromotor responses to spaceflight, which we have previously referred to as a gravitational unloading syndrome (3).
BGH IS RELEASED FOLLOWING NERVE STIMULATION IN RATS
The consistent reduction in the ability of rat pituitary glands to secrete BGH after spaceflight (9) is consistent with the idea that changes in proprioceptive stimuli associated with microgravity (3) may affect the regulation of BGH release. Therefore, we designed experiments to determine whether pituitary BGH secretion could be modulated by afferent input from skeletal muscle. Hindlimb nerves (ie, tibial, peroneal, soleus, or sural) were isolated surgically, severed, and electrically stimulated in anesthetized adult, male rats (5,6). To determine if BGH release could be elicited in response to a physiological level of afferent activation, the stimulation pattern simulated the electromyographic (EMG) activity recorded from rats running at 40 m·min−1 on a treadmill (14).
Stimulation of the proximal trunk of the tibial or peroneal nerve, innervating muscles composed of predominantly fast fibers, induced an ∼300% rise in plasma, and concurrent ∼50% decrease in pituitary, BGH concentrations (Figure 1) (6). The stimulation intensity excited only low threshold, large diameter (Group I and II) afferent fibers (5), indicating that muscle proprioceptors, spindles, and tendon organs were the source of the afferent input modulating BGH secretion. Although the duration of electrical stimulation was 15 min, near-maximal BGH responses occurred within 5–10 min, demonstrating the rapid onset at which the BGH secretory pathway or pathways were activated.
In contrast to stimulation of the tibial or peroneal nerve, stimulation of the proximal end of the severed soleus nerve, innervating the predominantly slow soleus muscle, inhibited BGH release (Figure 1) (5). It appears that only the BGH secretion, and not pituitary synthesis, was inhibited because plasma BGH decreased whereas pituitary BGH increased. The inhibition of BGH secretion after proximal soleus nerve stimulation suggested that alternate regulatory pathways from fast versus slow muscles exist for afferent modulation of BGH secretion. In addition, the stimulatory afferent inputs from fast muscles apparently can override the inhibitory inputs from slow muscles because BGH release occurred after stimulation of the proximal tibial nerve, which includes the soleus nerve trunk as well as nerves innervating several predominantly fast ankle extensor muscles.
BGH release was not affected by stimulation of the distal trunk of any nerve (Figure 1), indicating that BGH is not modulated by muscle contraction itself nor by any associated metabolic changes (6). There were no changes in other plasma metabolite or hormone concentrations, eg lactate, glucose, thyroxine, triiodothyronine, and testosterone, indicating they did not play a role in modulating BGH release (6). Moreover, stimulation of the proximal sural nerve had no effect on BGH secretion (Figure 1), demonstrating that the afferents that modulated BGH originated from skeletal muscle proprioceptors and not from cutaneous sources. No consistent changes in plasma or pituitary IGH concentrations occurred in response to any of the nerve stimulation paradigms.
The results from these nerve stimulation experiments clearly demonstrate that pituitary BGH release is induced by physiological levels of afferent input from hindlimb nerves innervating predominantly fast muscles of either the flexor or extensor compartments or inhibited by input from a slow extensor muscle. To our knowledge, this is the first evidence for a muscle-neuro-endocrine axis for modulating the release of an anterior pituitary growth factor via proprioceptive input.
EXERCISE-INDUCED BGH RELEASE
The results from in situ nerve stimulation in rats warranted further study of a comparable in vivo paradigm. Therefore, the BGH response to one acute bout of treadmill exercise was investigated (1). After a 15 min bout of running at 27 m·min−1, plasma BGH increased ∼300% and pituitary BGH decreased by ∼50% compared to nonexercised treadmill-acclimated or unacclimated control rats (Figure 2). Plasma and pituitary IGH concentrations were not changed by the acute bout of exercise. As with nerve stimulation, BGH release during the exercise paradigm was rapid and not metabolically or hormonally induced as evidenced by the absence of changes in plasma glucose, lactate, triglycerides, triiodothyronine, or testosterone levels. Given that a moderate speed of locomotion is probably accomplished by a large proportion of slow motor units as well as some fast motor units, any inhibitory effects on BGH release from afferent activity of predominantly slow muscle such as the soleus was apparently overridden by the activation of other muscles. In this regard, the exercise-induced BGH release is more analogous to afferent stimulation of the tibial nerve, which included both fast (plantaris, gastrocnemius) and slow (soleus) ankle extensor muscles. Whereas the threshold level of neuromuscular activity required for BGH release has yet to be determined, these data show that one moderate bout of running in untrained rats rapidly and markedly increases plasma and decreases pituitary BGH concentrations.
Figure 3A depicts the inverse relationship between plasma and pituitary BGH concentrations in rats over the range of experiments discussed above. Conditions that stimulate BGH release, such as fast muscle afferent activation and exercise, result in a concomitant increase in plasma and decrease in pituitary BGH, whereas inhibitory afferent input from the soleus muscle results in elevated pituitary and lowered plasma BGH. Furthermore, the pituitary and plasma BGH levels are similar among control, nonexercised, and cutaneous and distal nerve-stimulated rats. These data provide strong evidence that BGH is derived from the pituitary and that its secretion into the plasma can be modulated by afferent stimuli from skeletal muscle. Moreover, BGH is undetectable in the plasma of hypophysectomized rats subjected to these same stimuli (not shown in figure), providing further support for the pituitary origin of BGH. Lastly, there is no relationship between pituitary and plasma IGH for the same stimuli, indicating that BGH can be modulated differentially and independently from IGH (Figure 3B).
We have performed several experiments showing that a brief series of isometric muscle contractions increase circulating BGH in humans (11,12). In these studies, the exercise stimulus consisted of unilateral plantarflexion at maximal and submaximal intensities with 4-s contractions interspersed with 1-s rest intervals. Plasma BGH increased after as little as five min of isometric contractions at a 30% maximal voluntary contraction (MVC) intensity. This exercise-induced BGH response was transient, and plasma concentrations were similar to baseline levels by 30 min postexercise. When a combined series of submaximal and maximal contractions were performed for a similar duration, plasma BGH was elevated two- to three-fold immediately postexercise (Figure 4). This rapid time course for elevating plasma BGH levels is consistent with skeletal muscle afferent modulation. No changes in plasma IGH were elicited by any of these brief exercise protocols. Whereas exercise-induced increases in plasma IGH typically peak during or after longer duration aerobic or resistance exercise that involves a relatively large muscle mass (10), plasma BGH increases are more rapid, transient, and can occur after activation of a relatively small muscle mass. Thus, BGH and IGH have different thresholds for release and temporal responses to exercise.
When the leg musculature is chronically unloaded, such as during bed rest or spaceflight, exercise-induced elevations in plasma BGH are absent (11,12). This apparent deficit in BGH secretory capacity is consistent with the diminished ability of rat pituitary cells to secrete BGH during or after exposure to chronic unloading or microgravity (9). The absence of an exercise-induced plasma BGH increase occurs after only two to three days exposure to bed rest (11) or spaceflight (12) and persists for up to 14 d of unloading (Figure 4). These changes occurred in spite of the maintenance of maximal torque output (12) and normal or increased chronic EMG activity of the plantarflexor musculature (2). Furthermore, preexercise plasma BGH and IGH concentrations were unchanged, indicating that circulating basal levels were not affected by unloading. Based on the nerve stimulation studies performed in rats (5,6), we hypothesized that muscle spindle afferent activity during contractions modulated the exercise-induced BGH release from the pituitary, thereby resulting in elevated plasma BGH concentrations. Furthermore, we propose that the changes in the BGH exercise responses during spaceflight or bed rest can be attributed to the chronic alterations in proprioceptive inputs that accompany exposure to microgravity and unloading (3).
MUSCLE SPINDLE ACTIVITY MODULATES PLASMA BGH IN HUMANS
To test the hypothesis that muscle spindle activity could modulate plasma BGH in humans, we selectively stimulated group Ia muscle spindle afferent firing in relaxed leg muscles by applying a vibration stimulus to the surface of the skin overlying the muscle belly (13). Immediately after vibrating the tibialis anterior muscle for 10 min, plasma BGH concentrations were elevated by 94% (Figure 5). In contrast, 10 min of vibration of the soleus muscle did not significantly affect circulating BGH, although there was an indication for a decrease in plasma BGH immediately after vibration. Plasma IGH levels were similar before and after muscle vibration.
The results from these vibration experiments are analogous to those from the rat nerve stimulation studies in which BGH release from the pituitary was inhibited after afferent stimulation of the soleus nerve (5) and increased after peroneal (dorsiflexor muscles including tibialis anterior, extensor digitorum longus, and peronei) nerve stimulation (6). However, because soleus muscle afferent activity did not stimulate BGH release in either rats or humans, the plasma BGH elevations in humans after voluntary muscle contractions apparently are not modulated by the soleus muscle, despite the considerable EMG activity in this muscle during the plantarflexion exercise (12). Further investigation is needed to determine whether spindle activation in the gastrocnemius muscle, the other major plantarflexor, can increase plasma BGH and therefore account for the elevation that occurs after active plantarflexor contractions in humans. Nevertheless, the vibration experiments are convincing evidence for BGH modulation by muscle spindle activity in humans and support the theory that disruption of the exercise-induced BGH release during spaceflight or bed rest is the result of chronically altered muscle afferent activity.
CURRENT AND FUTURE DIRECTIONS
Physiology of BGH
The combined results from these experiments in rats and humans are consistent with the hypothesis that BGH secretion can be modulated by afferent input from skeletal muscle proprioceptors. Initial results in rats indicate that short periods of vibration of isolated tendons from fast hindlimb ankle extensors elicits BGH release similar to that seen following proximal nerve stimulation. In addition, the loss of this BGH secretion response following chronic periods of musculoskeletal unloading is being tested in rats by use of a ground-based hindlimb suspension model. Preliminary data indicate that hindlimb suspension decreases basal plasma BGH levels and abolishes the BGH secretory response to afferent nerve stimulation. Further studies are needed to determine the minimum level of activity required to elicit BGH secretion and to maintain the functional pathway or pathways during chronic unloading. Lastly, studies to determine whether BGH responds to aerobic exercise in humans are warranted because it is unknown whether BGH is secreted during this mode of exercise. Moreover, such human studies would be helpful in defining the mechanisms of BGH modulation as well as contrasting the temporal differences between BGH and the well-characterized IGH responses to exercise.
Characterization of BGH
Whereas our experiments to date have focused on the physiological mechanisms that modulate BGH release, characterization of the molecular structure of BGH will pave the way to determining what other physiological functions can be attributed to BGH in addition to longitudinal bone growth. Early studies on fractionated human and rat plasma showed that most of the BGH activity resided in the 60–80 kDa range (4). More recently, a 5 kDa peptide, termed “tibial peptide”, was isolated from human pituitary glands and shown to have activity in the tibial assay (8). Presently, we are attempting to determine the chemical nature of the BGH activity in plasma and primary rat pituitary culture media. Once this is known, a long list of questions need to be answered to more fully understand the physiological effects of BGH and contrast it with the more well-known effects of IGH. For example, does the BGH secreted from rat pituitary glands reside in multiple molecular weight fractions like IGH? Does BGH bind to carrier proteins for transport, stability, or modulation of its activity?
Development of an in vitro bioassay for BGH
The tibial bioassay is specific for GH in that other hormones in their range of physiological concentrations do not stimulate epiphyseal widening or interact with GH. This assay is an established and reproducible method for measuring the GH-like growth-promoting, ie BGH activity in biological samples such as plasma, pituitary tissue, or culture media. However, the assay is costly and requires large sample volumes. Therefore, we also are developing an in vitro cell-based assay, in which BGH appears to induce proliferation in a clonal rat chondrogenic cell line. Ongoing experiments will determine the effects of BGH and other hormones on these cells and whether this cell line will serve as an appropriate substitute for the tibial bioassay.
SUMMARY AND PERSPECTIVE
Multiple lines of indirect evidence support the existence of a muscle afferent–pituitary axis that can modulate the secretion of an uncharacterized pituitary growth factor historically referred to as BGH (Figure 6). This modulation of BGH release is muscle-specific: afferent activity from the tibialis anterior muscle stimulates BGH release, whereas that from the soleus muscle inhibits BGH release. However, the inhibitory effects of the soleus muscle can be overridden when accompanied by either nerve-stimulated or voluntary (exercise) afferent activity of additional plantar and dorsiflexor muscles. In rats, this differential response appeared to be related to the fiber type composition of the muscle, because the soleus muscle is predominantly composed of slow fibers in contrast to the predominantly fast fiber composition of the muscles that stimulated BGH release. This stimulating effect of fast muscles and inhibitory effect of a slow muscle in rats is paradoxical: the modulation seems to be derived from muscle spindles, but slow muscles have a higher spindle density than fast muscles. In humans, however, an increase in afferent activity (via vibration) of the tibialis anterior, a predominantly slow muscle in humans, increased plasma BGH. This may indicate that the regulation of BGH is more tightly coupled to muscle function rather than fiber type composition. Further research is needed to elucidate the functional significance of the muscle-specific regulation of BGH among and within species.
The BGH molecule that induces tibial growth remains to be characterized, and its novelty as a molecule is unknown. The range of biological effects of this proprioceptive-modulated growth factor remain to be determined, because almost all studies have focused on longitudinal bone growth. A number of physiological stimuli have been reported to disrupt BGH secretion independent of IGH, although the specific details leading to the altered BGH release have not been defined. The depression of activity-induced BGH secretion associated with periods of chronic absence of normal ambulation in a 1 g environment contributes to the enigma of the physiological regulatory processes. Is this depressed secretory response a factor in the adaptive response of bone, muscle, and other tissues to chronic bed rest or prolonged microgravity? To what extent has this regulatory function evolved such that this triggering mechanism has become dependent on some minimal level of ambulation in a 1 g environment? What role might altered BGH secretion play in the causation of chronic decreased activity-related diseases in general? Are there other physiological stimuli that modulate BGH secretion? The answers to these and other questions will help define the role of this unique muscle-neuro-endocrine axis in sustaining normal physiological function.
These studies were supported, in part, by National Institute of Neurological Disorders and Stroke Grant NS-16333 (VRE and RRR), National Aeronautic and Space Administration (NASA) Grant 199–26–12–09 (VRE, REG, and RRR), and NASA Space Physiology Research Grant from the American College of Sports Medicine (GEM). GEM and KLG were supported, in part, by a pre-doctoral training grant (National Research Service Award) from the National Institute of Dental Research (Grant DE-07212). KLG and AJB were supported, in part, by NASA Graduate Student Researchers Program pre-doctoral fellowships GSRP-98–104 and NGT2–52265, respectively. We are grateful to Dr. Jane E. Aubin of the University of Toronto for providing the clonal chondrogenic cell line.
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