Control of Anterior Pituitary Hormone Secretion by Neurotensin S. M. McCANNa AND E. VIJAYANb Department of Physiology, Neuropeptide Division f i e University of Ems Southwestern Medical Center at Dallas Dallas, Texas 75235-9040
INTRODUCTION In the rat, neurotensin is widely distributed in the central nervous system, particularly in limbic system structures. Cell bodies are found in the nucleus interstialis stria terminalis, medial preoptic area, periventricular region, paraventricular nucleus, arcuate nucleus, and lateral hypothalamus, areas that also contain fibers of these neurons. Some of these axons, particularly those arising in the arcuate nucleus, project to the lateral aspect of the external layer of the median eminence.'-3 Neurotensin may be colocalized with dopamine4.5 and CRF.6 This distribution clearly suggests an important role for the peptide in hypothalamic-pituitary function. Such a role is further supported by the presence of binding sites for the peptide in a variety of brain sites, including those related to areas where terminals or perikarya of neurotensin neurons are found.7 Anterior pituitary cells have been shown to contain neurotensin by immunocytochemistry.8 Levels of neurotensin in hypophyseal portal blood, collected from anesthetized rats by a modification of the technique of Worthington and Fink, were higher than those in peripheral blood, indicating that the peptide is secreted from the median eminence into portal blood and passes to the anterior pituitary.9 There, it may well have direct actions to alter pituitary hormone release. Since the hormone persists in the gland after pituitary stalk section, it not only reaches the pituitary via the portal vessels but also may actually be synthesized within pituitary cells.10 This is supported by the presence of mRNA for neurotensin in the pituitary gland, which was increased by ovariectomy and reversed by estrogen replacement therapy. 11 In fact, considerable evidence has now accumulated to indicate that the peptide can alter the release of most anterior pituitary hormones by hypothalamic and/or direct pituitary effects. The remainder of this chapter will be devoted to examining its effects on the various pituitary hormones, determining whether the actions are on the brain or the pituitary directly, evaluating the possible role of other neuronal systems in mediating the effects, evaluating the mechanism of action of the effects, and determining their physiological significance.
Address for correspondence: Department of Physiology, Neuropeptide Division, The University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 752359040, Present address: Department of Biological Sciences, Pondicherry University, Pondicherry-605004, India.
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ANNALS NEW YORK ACADEMY OF SCIENCES
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ROLE OF NEUROTENSIN IN CONTROL OF ANTERIOR PITUITARY HORMONE RELEASE Prolactin Hypothalamic Action The largest amount of work has been directed toward clarifying the role of neurotensin in the control of prolactin release. Following injection into the third ventricle (W), neurotensin can reduce plasma levels of prolactin in conscious ovariectomized, ovariectomized estrogen progesterone-blocked (OEP), or male rats. l2 It was difficult to observe this lowering of prolactin in conscious male rats because of the already low levels of prolactin; however, when these levels were elevated by exposing the animals to ether, a dramatic lowering of plasma prolactin was induced by intraventricular injection of neurotensin.13 Consequently, it is clear that the action of neurotensin on structures adjacent to the ventricle is to alter the balance of prolactin-releasing and prolactin-inhibiting factor release so as to suppress the release of prolactin from the adenohypophysis. Next, an attempt was made to determine which neurotransmitters might be involved in inducing the suppressive action of neurotensin. Fluoxetine, a blocker of reuptake of serotonin (5-hydroxytryptamine, 5HT) and 5-hydroxytryptophane (SHTP), a precursor of 5HT, produced marked elevations in plasma prolactin, presumably by increasing the amounts of 5HT in the synaptic clefts adjacent to the hypothalamic mechanisms controlling prolactin release. This elevation was dramatically reduced by intraventricular injection of neurotensin, which indicates that the suppressive action of neurotensin on prolactin release is not mediated directly by 5HT.13 On the other hand, when adrenergic transmission was blocked by the inhibitor of tyrosine hydroxylase, a-methyl para-tyrosine (aMT), to block catecholamine synthesis, prolactin levels also rose by elimination of catecholaminergic inhibitory control. Under these circumstances, the prolactin-lowering ability of intraventricular neurotensin was blocked. This was true whether or not plasma prolactin was elevated even further by ether anesthesia in the aMT-treated animals. These results indicate that the effect of neurotensin is mediated by catecholamines, which bring about the prolactinlowering action of the peptide, but do not identify the catecholamine involved. Spiroperidol, the dopamine receptor blocker, elevates plasma prolactin by elimination of dopaminergic inhibitory control; and in these animals neurotensin had no effect on plasma prolactin, which indicates that the catecholamine involved is dopamine. 13 The evidence indicates that intraventricular neurotensin stimulates the release of dopamine from dopaminergic neurons, presumably those of the tuberoinfundibular dopaminergic system. This dopamine then enters hypophyseal portal vessels and passes down to the pituitary to suppress prolactin release from the lactotrophs.I3 In vitro incubation experiments with hypothalamic fragments indicate that neurotensin can indeed release dopamine, so this mechanism appears to be fairly well established. I2+l4 Direct Pituitary Action In contrast to the prolactin-lowering activity of intraventricularly injected neurotensin, prolactin levels in plasma can be elevated by intravenous injection of the pep-
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tide. This action is almost certainly due to a direct stimulatory effect of the peptide on the anterior pituitary gland since in vitro incubation of hemipituitaries from ovariectomized female rats with neurotensin produced a dose-related stimulation of prolactin release. '* The direct stimulatory effect of neurotensin on the lactotrophs has been amply confirmed in subsequent work.15 The mechanism of stimulation appears to involve membrane polyphosphoinositide degradation. The inositol triphosphate (IP3) released liberates Ca2+ from intracellular stores. Diacylglycerol (DAG) is also released, which activates protein kinase C. This acts synergistically with the liberated Caz+.DAG as well as IPz, by the action of phospholipase Az, liberates archidonate which is metabolized to leukotrienes, prostaglandins, and epoxides which in turn may also stimulate prolactin release.I5
Physiological Role of Neurotensin in Altering Prolactin Release To examine the possible role of endogenous neurotensin to alter prolactin release, highly specific antiserum to neurotensin (NT-AS) was injected either into the third ventricle or intravenously into unanesthetized intact male, ovariectomized female or OEP rats. Intraventricular injection of 1 or 3 pl of NT-AS significantly increased plasma prolactin levels in ovariectomized as well as OEP rats on the first measurement one hour after injection, and the effect was dose-related at early times after the injection. In intact male rats intraventricular (3V) injection of NT-AS also produced an elevation of plasma prolactin, but the elevations were lower than those seen in ovariectomized or OEP rats. These results are opposite to those obtained from injection of the peptide itself and support the physiological significance of the intrahypothalamic inhibitory action of neurotensin in all these types of rats. Furthermore, they strongly suggest that neurotensin is involved in tonically controlling the basal secretion of prolactin. On the other hand, intravenous injection of a dose of antiserum, which had been previously shown to block the hypotensive effect of neurotensin, produced the opposite result and suppressed plasma prolactin concentrations in ovariectomized as well as OEP animals. The effect was more pronounced in OEP rats that have elevated plasma prolactin levels compared to their ovariectomized controls. Intravenous injection of the antiserum in intact males had no effect. The direct stimulatory action of the peptide on prolactin release appears to be physiologically and tonically significant in females. Apparently in the male the direct action of the peptide is not being exerted under resting conditions. The lack of physiological significance of neurotensin at the pituitary level in the male is also supported by the failure of the antiserum to alter prolactin release from dispersed anterior pituitary cells of males. Whether it would be demonstrable under conditions of enhanced release of prolactin requires further experimentation. l6 Thus, the evidence is extremely good that neurotensin has a suppressive action of physiological significance on the release of prolactin via the hypothalamus that is probably mediated by release of dopamine, which passes down the portal vessels and directly inhibits the lactotrophs. On the other hand, there is an opposite stimulatory action of physiological significance directly on the lactotrophs at least in the female. This situation is quite similar to that of many brain peptides, which appear to have opposite actions at hypothalamic and pituitary levels to alter pituitary hormone release.
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ANNALS NEW YORK ACADEMY OF SCIENCES
Follicle-Stimulating Hormone and Luteinizing Hormone In ovariectomized animals in which plasma follicle-stimulating hormone (FSH) and luteinizing hormone (LH) values were elevated because of removal of ovarian steroid negative feedback, intraventricular injection of neurotensin lowered plasma LH within 5 min and the effect persisted for 30 min after the 0.5-pg dose, but a progressive lowering occurred throughout the one-hour course of the experiment after the 2-pg dose. That this was a central effect is suggested by the fact that a dose of 1 pg given intravenously had no effect on plasma LH. In contrast to the effect on LH, neurotensin had no effect on plasma FSH. Since the peptide had no effect on the release of LH by hemipituitaries incubated in vitro, it was concluded that it exerted an inhibitory action on LHRH release in the castrate rat.12 Subsequently, Ferris et al. 17 microinjected neurotensin into the medial preoptic area of anesthetized, ovariectomized animals and found an elevation instead of a suppression of plasma LH but found, as we had, that injecting it into the ventricle lowered plasma LH levels. These observations suggest that the action of the peptide may be different depending on the locus in the hypothalamus affected. To determine the physiological significance of the effects of neurotensin on LH release, we injected purified neurotensin antiserum into the third ventricle of ovariectomized rats. The control injection of normal rabbit serum had no effect; however, the microinjection of 1 pl of neurotensin antiserum produced a slight increase in plasma LH at one hour after injection. Values remained elevated but not significantly so during the remaining four hours of observation. When the dose of antiserum was raised to 3 p1, a highly significant increase in plasma LH occurred, peaking at two and three hours, which was followed by a precipitous fall back to control values at four and five hours after injection. This observation supports the physiological significance of the plasma LH lowering effect that occurred after intraventricular injection of the peptide. That the action of the neurotensin antiserum was exerted within the brain is suggested by the fact that there was no effect of peripheral injection of the antiserum in ovariectomized animals just as there had been no effect of i.v. injection of the peptide itself (Vijayan et al., unpublished data, 1988). The plasma LH levels were elevated after intraventricular injection of the antiserum in ovariectomized, estrogen progesterone-primed animals at three, four, and five hours after the intraventricular injection of the lower but not the higher dose. This supports the physiological significance of the inhibitory action of endogenous neurotensin in this type of rat as well (Vijayan et al., unpublished, 1988). Recently, Alexander et al. 18 microinjected antiserum against neurotensin into the medial preoptic area in the afternoon in ovariectomized, estrogen progesterone-treated animals that were undergoing a proestrus-like surge of LH. The antiserum blocked the LH surge, indicating that the stirnulatory action of the peptide in the preoptic area found earlier by this group may have physiological significance in augmenting the proestrous surge of LH, which further supports the concept of opposite actions of the peptide at rostral and more caudal sites in the hypothalamus. Perhaps the failure of the higher intraventricular dose of NT-AS to affect plasma LH in our experiments may be related to spread of the antiserum from the area in which neurotensin inhibits LHRH release to the area in which it stimulates it. This stirnulatory action of neurotensin on LHRH release in the estrogen-primed rat appears to be mediated via dopamine since a dopamine receptor blocker, pimozide,
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can block the elevation of LH produced by injection of neurotensin into the medial preoptic area of estrogen-primed rats. l9 It is now apparent that blockade of the action of a number of transmitters or neuromodulators can interfere with the proestrous surge of LH. Blockade of a-adrenergic, dopaminergic,20 neuropeptide Y,2' angiotensin 11,22and oxytocin23transmission as well as blockade of the release or action of LHRH blocks the surge.20 Furthermore, LHRH apparently exerts a positive feedback at this time to augment its own release during the surge.24Thus, the proestrous surge of LHRH is probably due to a stimulatory action of a variety of factors. The LH surge may be further augmented by direct action on the pituitary of transmitters released into the hypophyseal portal vessels at this time, which could include not only LHRH, but also neuropeptide Y25and even epinephrine and norepinephrine.26Additionally, the gonadotropes are supersensitive to LHRH via the action of estrogen and the self-priming action of LHRH further amplifies the surge.20
Follicle-Stimulating Hormone In contrast to the dramatic effects on LH release following the third ventricular injection of the peptide itself or the antisera directed to it, there was little effect on plasma FSH under any of the conditions described above for LH. This provides another example of dissociation in the release of FSH and LH and strongly suggests the separate hypothalamic control of these two gonadotropins. Many other examples of dissociation in the release of FSH and LH have been described. It is believed that the hypothalamic control of FSH is at least in part under the control of an FSH-releasing factor. Cell bodies of this factor are postulated to exist in the region of the periventricular nucleus with axons projecting particularly to the caudal median eminence. The factor has been partially purified, and synthetic peptides have been shown to possess selective FSH-releasing activity.27.28
Thyroid-Stimulating Hormone Intraventricular injection of either 0.5 or 2 pg of neurotensin failed to modify plasma levels of thyroid-stimulating hormone (TSH); however, intravenous injection of 1 pg of the peptide dramatically elevated plasma TSH within 5 min, and this effect persisted for 30 min after injection. The effect of the intravenous injection of neurotensin was probably mediated at the pituitary level, since incubation of hemipituitaries from ovariectomized animals revealed a stimulatory effect of neurotensin on TSH release at a minimal effective dose of 4 nM of the peptide. There was a suggestion of a doseresponse relationship.29 This pituitary action of neurotensin may have physiological significance since intravenous injection of neurotensin antiserum lowered plasma TSH in both ovariectomized and ovariectomized, estrogen progesterone-treated rats. A suppression of plasma TSH also occurred after intraventricular injection of the antiserum in males as well as the ovariectomized female groups. Since intraventricular injection of the peptide itself had no effect, these effects may be related to spread of the antiserum to the pituitary with suppression of the stimulation of TSH release induced by endogenous neurotensin (Vijayan et al., unpublished, 1988).
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Growth Hormone Third ventricular injection of 2 pg of neurotensin induced dramatic elevations in plasma growth hormone within 5 min of injection, and values remained significantly elevated for the 60-minute duration of the experiment. They were also elevated following injection of a lower, 0.5-pg dose, but declined to control values within 60 min. There was no significant difference between the response to the two doses except at this later time. When hemipituitaries of ovariectomized animals were incubated with the peptide, there was no effect on growth hormone release, indicating that the actions observed after intraventricular injection were mediated at the hypothalamic level .29 In the experiments with the intraventricular injection of neurotensin antiserum, it was found that the antiserum lowered growth hormone in ovariectomized females, OEP females, and also male rats. Thus, the peptide appears to have a physiologically significantaction within the hypothalamus to stimulate growth hormone secretion either via activation of growth hormone-releasing factor release, inhibition of somatostatin release, or by both actions (Vijayan et af., unpublished, 1989). Surprisingly, the intravenousinjection of the antiserum produced the opposite effect and caused an elevation in growth hormone levels in both types of ovariectomized rats. We have no explanation for this latter result since we found no effect of the peptide on growth hormone release in vitro. Perhaps there is a direct inhibitory effect on the release of growth hormone that went undetected.
DISCUSSION Neurotensin has important effects that alter pituitary hormone secretion by both hypothalamic and pituitary actions. It is clear that the peptide suppresses prolactin release by stimulation of the release of dopamine, which then directly inhibits secretion at the lactotrophs. This effect appears to be of physiologic significance based on the antiserum studies (TABLE1). In addition, there is a direct stirnulatory effect of neurotensin on prolactin release, and this is of physiologic significance at least in the female (FIG.1). In the case of LH there may be dual actions of the peptide that stimulate the release of luteinizing hormone releasing hormone (LHRH) via a dopaminergic step in the medial preoptic area and inhibit the release of LHRH more caudally by actions on
TABLE1. Summary of Results with Neurotensin Antiserum Type of Rat
Route of Injection
Effect on Plasma Hormone Concentration
FSH
LH
PrI
GH
TSH
+ +
-
-
Male
3v
0
0
ovx
3v IV
0 0
+
-
-
-
0
+
-
3v
0
+
+
-
-
N
0
0
OEP
-
+
-
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NT n.
LACTOTROPE FIGURE 1. The diagrammatic illustration of the role of neurotensin in control of prolactin release. 2: OC = optic chiasm; MB = marnmallary bodies; ME Key to abbreviations in this and FIGURE = median eminence; PV = portal vessel; PP = posterior pituitary; AP = anterior pituitary; NT n. = neurotensin neuron; DA = dopamine. + = stimulation; - = inhibition.
the median eminence LHRH neuronal axons or interneurons connected to these. Both of these actions appear to be of physiologic significance based on the antibody studies. There appears to be no direct action of the peptide on the gonadotrophs (FIG. 2). As in the case of many other brain peptides, although there are clear effects of the peptide on LHRH release, there is little or no effect on FSH release, providing yet another example of dissociation of the hypothalamic control of these two gonadotropin~.*~
INTRODUCTION In the rat, neurotensin is widely distributed in the central nervous system, particularly in limbic system structures. Cell bodies are found in the nucleus interstialis stria terminalis, medial preoptic area, periventricular region, paraventricular nucleus, arcuate nucleus, and lateral hypothalamus, areas that also contain fibers of these neurons. Some of these axons, particularly those arising in the arcuate nucleus, project to the lateral aspect of the external layer of the median eminence.'-3 Neurotensin may be colocalized with dopamine4.5 and CRF.6 This distribution clearly suggests an important role for the peptide in hypothalamic-pituitary function. Such a role is further supported by the presence of binding sites for the peptide in a variety of brain sites, including those related to areas where terminals or perikarya of neurotensin neurons are found.7 Anterior pituitary cells have been shown to contain neurotensin by immunocytochemistry.8 Levels of neurotensin in hypophyseal portal blood, collected from anesthetized rats by a modification of the technique of Worthington and Fink, were higher than those in peripheral blood, indicating that the peptide is secreted from the median eminence into portal blood and passes to the anterior pituitary.9 There, it may well have direct actions to alter pituitary hormone release. Since the hormone persists in the gland after pituitary stalk section, it not only reaches the pituitary via the portal vessels but also may actually be synthesized within pituitary cells.10 This is supported by the presence of mRNA for neurotensin in the pituitary gland, which was increased by ovariectomy and reversed by estrogen replacement therapy. 11 In fact, considerable evidence has now accumulated to indicate that the peptide can alter the release of most anterior pituitary hormones by hypothalamic and/or direct pituitary effects. The remainder of this chapter will be devoted to examining its effects on the various pituitary hormones, determining whether the actions are on the brain or the pituitary directly, evaluating the possible role of other neuronal systems in mediating the effects, evaluating the mechanism of action of the effects, and determining their physiological significance.
Address for correspondence: Department of Physiology, Neuropeptide Division, The University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 752359040, Present address: Department of Biological Sciences, Pondicherry University, Pondicherry-605004, India.
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ANNALS NEW YORK ACADEMY OF SCIENCES
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ROLE OF NEUROTENSIN IN CONTROL OF ANTERIOR PITUITARY HORMONE RELEASE Prolactin Hypothalamic Action The largest amount of work has been directed toward clarifying the role of neurotensin in the control of prolactin release. Following injection into the third ventricle (W), neurotensin can reduce plasma levels of prolactin in conscious ovariectomized, ovariectomized estrogen progesterone-blocked (OEP), or male rats. l2 It was difficult to observe this lowering of prolactin in conscious male rats because of the already low levels of prolactin; however, when these levels were elevated by exposing the animals to ether, a dramatic lowering of plasma prolactin was induced by intraventricular injection of neurotensin.13 Consequently, it is clear that the action of neurotensin on structures adjacent to the ventricle is to alter the balance of prolactin-releasing and prolactin-inhibiting factor release so as to suppress the release of prolactin from the adenohypophysis. Next, an attempt was made to determine which neurotransmitters might be involved in inducing the suppressive action of neurotensin. Fluoxetine, a blocker of reuptake of serotonin (5-hydroxytryptamine, 5HT) and 5-hydroxytryptophane (SHTP), a precursor of 5HT, produced marked elevations in plasma prolactin, presumably by increasing the amounts of 5HT in the synaptic clefts adjacent to the hypothalamic mechanisms controlling prolactin release. This elevation was dramatically reduced by intraventricular injection of neurotensin, which indicates that the suppressive action of neurotensin on prolactin release is not mediated directly by 5HT.13 On the other hand, when adrenergic transmission was blocked by the inhibitor of tyrosine hydroxylase, a-methyl para-tyrosine (aMT), to block catecholamine synthesis, prolactin levels also rose by elimination of catecholaminergic inhibitory control. Under these circumstances, the prolactin-lowering ability of intraventricular neurotensin was blocked. This was true whether or not plasma prolactin was elevated even further by ether anesthesia in the aMT-treated animals. These results indicate that the effect of neurotensin is mediated by catecholamines, which bring about the prolactinlowering action of the peptide, but do not identify the catecholamine involved. Spiroperidol, the dopamine receptor blocker, elevates plasma prolactin by elimination of dopaminergic inhibitory control; and in these animals neurotensin had no effect on plasma prolactin, which indicates that the catecholamine involved is dopamine. 13 The evidence indicates that intraventricular neurotensin stimulates the release of dopamine from dopaminergic neurons, presumably those of the tuberoinfundibular dopaminergic system. This dopamine then enters hypophyseal portal vessels and passes down to the pituitary to suppress prolactin release from the lactotrophs.I3 In vitro incubation experiments with hypothalamic fragments indicate that neurotensin can indeed release dopamine, so this mechanism appears to be fairly well established. I2+l4 Direct Pituitary Action In contrast to the prolactin-lowering activity of intraventricularly injected neurotensin, prolactin levels in plasma can be elevated by intravenous injection of the pep-
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tide. This action is almost certainly due to a direct stimulatory effect of the peptide on the anterior pituitary gland since in vitro incubation of hemipituitaries from ovariectomized female rats with neurotensin produced a dose-related stimulation of prolactin release. '* The direct stimulatory effect of neurotensin on the lactotrophs has been amply confirmed in subsequent work.15 The mechanism of stimulation appears to involve membrane polyphosphoinositide degradation. The inositol triphosphate (IP3) released liberates Ca2+ from intracellular stores. Diacylglycerol (DAG) is also released, which activates protein kinase C. This acts synergistically with the liberated Caz+.DAG as well as IPz, by the action of phospholipase Az, liberates archidonate which is metabolized to leukotrienes, prostaglandins, and epoxides which in turn may also stimulate prolactin release.I5
Physiological Role of Neurotensin in Altering Prolactin Release To examine the possible role of endogenous neurotensin to alter prolactin release, highly specific antiserum to neurotensin (NT-AS) was injected either into the third ventricle or intravenously into unanesthetized intact male, ovariectomized female or OEP rats. Intraventricular injection of 1 or 3 pl of NT-AS significantly increased plasma prolactin levels in ovariectomized as well as OEP rats on the first measurement one hour after injection, and the effect was dose-related at early times after the injection. In intact male rats intraventricular (3V) injection of NT-AS also produced an elevation of plasma prolactin, but the elevations were lower than those seen in ovariectomized or OEP rats. These results are opposite to those obtained from injection of the peptide itself and support the physiological significance of the intrahypothalamic inhibitory action of neurotensin in all these types of rats. Furthermore, they strongly suggest that neurotensin is involved in tonically controlling the basal secretion of prolactin. On the other hand, intravenous injection of a dose of antiserum, which had been previously shown to block the hypotensive effect of neurotensin, produced the opposite result and suppressed plasma prolactin concentrations in ovariectomized as well as OEP animals. The effect was more pronounced in OEP rats that have elevated plasma prolactin levels compared to their ovariectomized controls. Intravenous injection of the antiserum in intact males had no effect. The direct stimulatory action of the peptide on prolactin release appears to be physiologically and tonically significant in females. Apparently in the male the direct action of the peptide is not being exerted under resting conditions. The lack of physiological significance of neurotensin at the pituitary level in the male is also supported by the failure of the antiserum to alter prolactin release from dispersed anterior pituitary cells of males. Whether it would be demonstrable under conditions of enhanced release of prolactin requires further experimentation. l6 Thus, the evidence is extremely good that neurotensin has a suppressive action of physiological significance on the release of prolactin via the hypothalamus that is probably mediated by release of dopamine, which passes down the portal vessels and directly inhibits the lactotrophs. On the other hand, there is an opposite stimulatory action of physiological significance directly on the lactotrophs at least in the female. This situation is quite similar to that of many brain peptides, which appear to have opposite actions at hypothalamic and pituitary levels to alter pituitary hormone release.
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ANNALS NEW YORK ACADEMY OF SCIENCES
Follicle-Stimulating Hormone and Luteinizing Hormone In ovariectomized animals in which plasma follicle-stimulating hormone (FSH) and luteinizing hormone (LH) values were elevated because of removal of ovarian steroid negative feedback, intraventricular injection of neurotensin lowered plasma LH within 5 min and the effect persisted for 30 min after the 0.5-pg dose, but a progressive lowering occurred throughout the one-hour course of the experiment after the 2-pg dose. That this was a central effect is suggested by the fact that a dose of 1 pg given intravenously had no effect on plasma LH. In contrast to the effect on LH, neurotensin had no effect on plasma FSH. Since the peptide had no effect on the release of LH by hemipituitaries incubated in vitro, it was concluded that it exerted an inhibitory action on LHRH release in the castrate rat.12 Subsequently, Ferris et al. 17 microinjected neurotensin into the medial preoptic area of anesthetized, ovariectomized animals and found an elevation instead of a suppression of plasma LH but found, as we had, that injecting it into the ventricle lowered plasma LH levels. These observations suggest that the action of the peptide may be different depending on the locus in the hypothalamus affected. To determine the physiological significance of the effects of neurotensin on LH release, we injected purified neurotensin antiserum into the third ventricle of ovariectomized rats. The control injection of normal rabbit serum had no effect; however, the microinjection of 1 pl of neurotensin antiserum produced a slight increase in plasma LH at one hour after injection. Values remained elevated but not significantly so during the remaining four hours of observation. When the dose of antiserum was raised to 3 p1, a highly significant increase in plasma LH occurred, peaking at two and three hours, which was followed by a precipitous fall back to control values at four and five hours after injection. This observation supports the physiological significance of the plasma LH lowering effect that occurred after intraventricular injection of the peptide. That the action of the neurotensin antiserum was exerted within the brain is suggested by the fact that there was no effect of peripheral injection of the antiserum in ovariectomized animals just as there had been no effect of i.v. injection of the peptide itself (Vijayan et al., unpublished data, 1988). The plasma LH levels were elevated after intraventricular injection of the antiserum in ovariectomized, estrogen progesterone-primed animals at three, four, and five hours after the intraventricular injection of the lower but not the higher dose. This supports the physiological significance of the inhibitory action of endogenous neurotensin in this type of rat as well (Vijayan et al., unpublished, 1988). Recently, Alexander et al. 18 microinjected antiserum against neurotensin into the medial preoptic area in the afternoon in ovariectomized, estrogen progesterone-treated animals that were undergoing a proestrus-like surge of LH. The antiserum blocked the LH surge, indicating that the stirnulatory action of the peptide in the preoptic area found earlier by this group may have physiological significance in augmenting the proestrous surge of LH, which further supports the concept of opposite actions of the peptide at rostral and more caudal sites in the hypothalamus. Perhaps the failure of the higher intraventricular dose of NT-AS to affect plasma LH in our experiments may be related to spread of the antiserum from the area in which neurotensin inhibits LHRH release to the area in which it stimulates it. This stirnulatory action of neurotensin on LHRH release in the estrogen-primed rat appears to be mediated via dopamine since a dopamine receptor blocker, pimozide,
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can block the elevation of LH produced by injection of neurotensin into the medial preoptic area of estrogen-primed rats. l9 It is now apparent that blockade of the action of a number of transmitters or neuromodulators can interfere with the proestrous surge of LH. Blockade of a-adrenergic, dopaminergic,20 neuropeptide Y,2' angiotensin 11,22and oxytocin23transmission as well as blockade of the release or action of LHRH blocks the surge.20 Furthermore, LHRH apparently exerts a positive feedback at this time to augment its own release during the surge.24Thus, the proestrous surge of LHRH is probably due to a stimulatory action of a variety of factors. The LH surge may be further augmented by direct action on the pituitary of transmitters released into the hypophyseal portal vessels at this time, which could include not only LHRH, but also neuropeptide Y25and even epinephrine and norepinephrine.26Additionally, the gonadotropes are supersensitive to LHRH via the action of estrogen and the self-priming action of LHRH further amplifies the surge.20
Follicle-Stimulating Hormone In contrast to the dramatic effects on LH release following the third ventricular injection of the peptide itself or the antisera directed to it, there was little effect on plasma FSH under any of the conditions described above for LH. This provides another example of dissociation in the release of FSH and LH and strongly suggests the separate hypothalamic control of these two gonadotropins. Many other examples of dissociation in the release of FSH and LH have been described. It is believed that the hypothalamic control of FSH is at least in part under the control of an FSH-releasing factor. Cell bodies of this factor are postulated to exist in the region of the periventricular nucleus with axons projecting particularly to the caudal median eminence. The factor has been partially purified, and synthetic peptides have been shown to possess selective FSH-releasing activity.27.28
Thyroid-Stimulating Hormone Intraventricular injection of either 0.5 or 2 pg of neurotensin failed to modify plasma levels of thyroid-stimulating hormone (TSH); however, intravenous injection of 1 pg of the peptide dramatically elevated plasma TSH within 5 min, and this effect persisted for 30 min after injection. The effect of the intravenous injection of neurotensin was probably mediated at the pituitary level, since incubation of hemipituitaries from ovariectomized animals revealed a stimulatory effect of neurotensin on TSH release at a minimal effective dose of 4 nM of the peptide. There was a suggestion of a doseresponse relationship.29 This pituitary action of neurotensin may have physiological significance since intravenous injection of neurotensin antiserum lowered plasma TSH in both ovariectomized and ovariectomized, estrogen progesterone-treated rats. A suppression of plasma TSH also occurred after intraventricular injection of the antiserum in males as well as the ovariectomized female groups. Since intraventricular injection of the peptide itself had no effect, these effects may be related to spread of the antiserum to the pituitary with suppression of the stimulation of TSH release induced by endogenous neurotensin (Vijayan et al., unpublished, 1988).
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ANNALS NEW YORK ACADEMY OF SCIENCES
Growth Hormone Third ventricular injection of 2 pg of neurotensin induced dramatic elevations in plasma growth hormone within 5 min of injection, and values remained significantly elevated for the 60-minute duration of the experiment. They were also elevated following injection of a lower, 0.5-pg dose, but declined to control values within 60 min. There was no significant difference between the response to the two doses except at this later time. When hemipituitaries of ovariectomized animals were incubated with the peptide, there was no effect on growth hormone release, indicating that the actions observed after intraventricular injection were mediated at the hypothalamic level .29 In the experiments with the intraventricular injection of neurotensin antiserum, it was found that the antiserum lowered growth hormone in ovariectomized females, OEP females, and also male rats. Thus, the peptide appears to have a physiologically significantaction within the hypothalamus to stimulate growth hormone secretion either via activation of growth hormone-releasing factor release, inhibition of somatostatin release, or by both actions (Vijayan et af., unpublished, 1989). Surprisingly, the intravenousinjection of the antiserum produced the opposite effect and caused an elevation in growth hormone levels in both types of ovariectomized rats. We have no explanation for this latter result since we found no effect of the peptide on growth hormone release in vitro. Perhaps there is a direct inhibitory effect on the release of growth hormone that went undetected.
DISCUSSION Neurotensin has important effects that alter pituitary hormone secretion by both hypothalamic and pituitary actions. It is clear that the peptide suppresses prolactin release by stimulation of the release of dopamine, which then directly inhibits secretion at the lactotrophs. This effect appears to be of physiologic significance based on the antiserum studies (TABLE1). In addition, there is a direct stirnulatory effect of neurotensin on prolactin release, and this is of physiologic significance at least in the female (FIG.1). In the case of LH there may be dual actions of the peptide that stimulate the release of luteinizing hormone releasing hormone (LHRH) via a dopaminergic step in the medial preoptic area and inhibit the release of LHRH more caudally by actions on
TABLE1. Summary of Results with Neurotensin Antiserum Type of Rat
Route of Injection
Effect on Plasma Hormone Concentration
FSH
LH
PrI
GH
TSH
+ +
-
-
Male
3v
0
0
ovx
3v IV
0 0
+
-
-
-
0
+
-
3v
0
+
+
-
-
N
0
0
OEP
-
+
-
McCANN & VIJAYAN: ANTERIOR PITUITARY SECRETION
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NT n.
LACTOTROPE FIGURE 1. The diagrammatic illustration of the role of neurotensin in control of prolactin release. 2: OC = optic chiasm; MB = marnmallary bodies; ME Key to abbreviations in this and FIGURE = median eminence; PV = portal vessel; PP = posterior pituitary; AP = anterior pituitary; NT n. = neurotensin neuron; DA = dopamine. + = stimulation; - = inhibition.
the median eminence LHRH neuronal axons or interneurons connected to these. Both of these actions appear to be of physiologic significance based on the antibody studies. There appears to be no direct action of the peptide on the gonadotrophs (FIG. 2). As in the case of many other brain peptides, although there are clear effects of the peptide on LHRH release, there is little or no effect on FSH release, providing yet another example of dissociation of the hypothalamic control of these two gonadotropin~.*~
