Ricin-cytotoxin conjugate administration reveals a physiologically relevant role for oxytocin in the control of gonadotropin secretion.
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Animals↗
Biomedical subjects
W K Samson
Publications and source records attributed to W K Samson.
A prolactin release inhibiting activity isolated from neurointermediate lobe extracts is an endothelin-like peptide.
A prolactin release-inhibiting factor has been reported in extracts of rat and bovine neurointermediate lobes of the pituitary gland. This material when isolated by Sephadex G-25 chromatography or by C-8 high pressure liquid chromatography coelutes with synthetic forms of all members of the mammalian endothelin family of peptides and with their immediate precursors, big-ET-1 and big-ET-3. The endogenous material crossreacts in an endothelin radioimmunoassay. Immunohistochemical analysis of endothelin-like immunoreactivity in the neurointermediate lobe revealed a predominantly intermediate lobe localization. Individual immunopositive cells were visualized in the intermediate lobe. Preabsorption of chromatographically isolated prolactin release-inhibiting activity with an endothelin antiserum which recognizes all members of the ET family of peptides abolishes its in vitro bioactivity. These results suggest that a prolactin release-inhibiting activity present in the neurointermediate lobe is endothelin or an endothelin-like peptide.
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Immunohistochemical evidence for the presence of oxytocin in the opossum corpus luteum.
Corpora lutea from opossums late in pregnancy were examined by immunohistochemistry for the presence of oxytocin. Oxytocin-immunoreactivity was observed in all corpora lutea examined but not elsewhere in ovarian tissue. The immunoreactive staining observed was confined primarily to the perinuclear cytoplasm of reactive luteal cells. Not all luteal cells showed oxytocin-immunoreactivity. The immunohistochemical localization of oxytocin in the pregnant opossum corpus luteum demonstrates for the first time this peptide in a metatherian ovary. Its presence in this primitive species suggests that oxytocin has a fundamental role in the physiology of the mammalian ovary.
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Central peptidergic mechanisms controlling reproductive hormone secretion: novel methodology reveals a role for the natriuretic peptides.
A variety of neural factors can influence reproductive hormone secretion by neuromodulatory actions within the hypothalamus or neuroendocrine actions within the anterior pituitary gland. Passive immunoneutralization and antagonist administration protocols have suggested physiological roles for a number of these factors; however, both experimental approaches have severe technical limitations. We have developed novel methodology utilizing cytotoxin cell targeting with neuropeptides linked to the toxic A chain of the plant cytotoxin ricin. With this methodology we can target and destroy in vivo or in vitro cells bearing receptors for that peptide. Ricin A chain conjugated to atrial natriuretic peptide (ANP), a neuropeptide known to pharmacologically inhibit luteinizing hormone-releasing hormone (LHRH) release, was injected into the cerebroventricular system of intact, cycling rats and ovariectomized rats. Cytotoxin conjugate treatment significantly lengthened the estrous cycle. In ovariectomized rats the luteinizing hormone surge induced by steroid priming was completely inhibited. LHRH content of the median eminences of these rats was not significantly altered. These data suggest that ANP binding to clearance receptors in the hypothalamus displaces the C-type natriuretic peptide (CNP) from the shared clearance receptor, making more CNP available to inhibit LHRH release. In the absence of cells bearing the clearance receptor all available CNP binds to the ANPR-B receptor and exerts its effect via an inhibitory interneuron, since LHRH fibers are spared by this treatment.
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Vasoconstrictor-induced secretion of ANP in fetal sheep.
Fetal secretion of atrial natriuretic peptide (ANP) increases during volume expansion and hypoxia. It is unknown whether this is associated with alterations in right atrial pressure (RAP) or distension and whether increases in ANP secretion reflect effects of specific vasopressors. To address this we studied fetal sheep (n = 13) at 125-140 days gestation, infusing either angiotensin II (ANG II; 0.023-5.73 micrograms/min) or the alpha-agonist phenylephrine (Phen; 0.031-7.64 micrograms/min) while monitoring mean arterial pressure (MAP), RAP, heart rate, and amniotic sac pressure. Arterial blood was obtained before and at 5 min of infusion to measure ANP, blood gases, and pH; umbilical venous blood was collected to determine placental clearance of ANP. ANG II caused dose-dependent increases in MAP and plasma ANP (P less than 0.05), whereas Phen caused dose-dependent increases in MAP, but ANP rose only with the highest dose (40 +/- 12%). delta MAP and delta RAP were highly correlated for Phen (r = 0.74, P = 0.002) and ANG II (r = 0.90, P less than 0.001), but for both agents the increase in delta RAP was proportionately greater than delta MAP, and increases in plasma ANP were greater per millimeter mercury rise in RAP than that observed with MAP. Increases in ANP were associated with a dose-dependent rise in hematocrit, suggesting decreases in intravascular volume. There was no fetal placental clearance of ANP. As in adults, ANG II- and alpha-agonist-induced fetal ANP secretion appears to primarily reflect increases in RAP and thus right atrial distension.
Angiotensin II↗
Comparison of the pituitary effects of the mammalian endothelins: vasoactive intestinal contractor (endothelin-beta, rat endothelin-2) is a potent inhibitor of prolactin secretion.
Direct pituitary effects of vasoactive intestinal contractor (VIC), which has been described recently to be the rat form of endothelin-2 (ET-2), were compared to those previously reported for rat ET-1, rat ET-3, and human ET-2. In static incubations of cultured dispersed anterior pituitary cells, the minimum effective dose of VIC necessary to inhibit PRL release after 1-h incubation was 1 pM, and the maximum effective dose was 1 nM. Similar inhibition was observed with human ET-2. The minimum effective inhibitory dose of ET-1 was also 1 pM; however, that of ET-3 was 0.1 nM. PRL release inhibition by VIC was not mediated via the D2-dopamine receptor and was not prevented by calcium channel blockade with 100 nM nifedipine. The inhibitory effect of VIC was not present in cells treated with 100 nM staurosporine, a dose that inhibits protein kinase-C activity. Time-course studies revealed a transient stimulation of PRL release with higher doses of VIC (10 and 100 nM), which occurred within the first 15 min of incubation and was unaffected by calcium channel blockade or inhibition of protein kinase-C activity. No stimulation of PRL release was observed with doses of VIC lower than 10 nM. Instead, we observed the maintenance of the inhibitory effect for 4 h of incubation. GH release was not significantly affected by doses of VIC ranging from 10(-13)-10(-7) M; however, the release of LH was slightly, yet significantly, stimulated by 10 and 100 nM VIC. This release was prevented by pretreatment with nifedipine, but unaffected by protein kinase-C inactivation. A physiological role for VIC (rat ET-2) in the control of lactotroph function is suggested by its effectiveness at picomolar doses and its long-lasting action.
Alkaloids↗
A highly sensitive and rapid radioimmunoassay for the determination of arginine8-vasopressin.
A highly sensitive radioimmunossay for arginine8-vasopressin (argipressin; INN) measurement was developed using Amberlite XAD 2 resin columns to extract arginine8-vasopressin from acidified human plasma. Arginine8-vasopressin was determined by a rapid radioimmunoassay method (2 x 20 h) using a specific antibody and 125I-labelled antigen. The bound fraction was separated by adsorption of the free fraction onto bovine serum albumin-coated charcoal; this resulted in low unspecific binding of less than 2%. Recovery experiments in the physiological range resulted in a mean (+/- SEM) recovery of 88 +/- 3%. The radioimmunoassay consistently yielded a detection limit of 0.3 ng/l (ED90) and a mean 50% binding intercept (ED50) of 3.5 ng/l. Arginine8-vasopressin immunoreactivity was characterized by reverse-phase high performance liquid chromatography, which confirmed the specificity of the assay. Serial plasma dilution curves paralleled the standard curve. The intra- and inter-assay variations were 9.4% and 15%, respectively. Arginine8-vasopressin concentrations in healthy subjects were determined in normal hydration status (2.2 +/- 0.3 ng/l; n = 11), as well as during suppression by water immersion (1.5 +/- 0.2 ng/l; n = 11) or by water loading (1.6 +/- 0.2 ng/l; n = 8). Thus, this assay allows for a sensitive, accurate and rapid quantification of plasma arginine8-vasopressin concentrations.
Arginine Vasopressin↗
CNP-22 stimulates, rather than inhibits, water drinking in the rat: evidence for a unique biological action of the C-type natriuretic peptides.
C-type natriuretic peptide (CNP) shares structural homology with A-type natriuretic peptide (ANP). Unlike ANP, which inhibits experimentally induced water drinking, CNP stimulates intake under similar conditions. The action of CNP to stimulate water drinking is not due to competition with ANP for the clearance receptor which recognizes both peptides since the ligand specific for that binding site, C-ANF 4-23, like ANP, inhibits water drinking under the same conditions.
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Central nervous system action of endothelin-3 to inhibit water drinking in the rat.
Endothelin-3, a vasoconstrictor peptide produced not only in endothelial cells but also within the brain interstitium in magnocellular neurons of the hypothalamo-neurohypophysial system, exerted significant, dose-related, inhibitory effects on water intake when administered into the third cerebroventricle of rats exposed to exogenous and endogenous stimuli for drinking. Passive immunoneutralization of endogenous endothelin-3 accentuated water drinking in response to angiotensin II administration suggesting a physiological role for the peptide in the central control of fluid and electrolyte homeostasis.
Angiotensin II↗
Possible neuroendocrine actions of endothelin-3.
The presence of endothelin (ET) immunoreactivity and binding sites in hypothalamus and pituitary gland suggests potential neuroendocrine actions of this family of vasoactive peptides. ET-3, the predominant member of the ET family in brain, exerted significant dose-related (1, 10, and 100 nM) inhibitory effects on PRL release from dispersed anterior pituitary cells in static incubations. The effect was not dependent on voltage-sensitive calcium channels, since the dihydropyridine calcium channel antagonist nifedipine failed to block this action. Nifedipine did, however, significantly reduce the transient acute stimulatory effect of ET-3 on PRL release in cultured cells incubated in dynamic perifusion. The longer lasting inhibitory effect on PRL release that followed the brief stimulatory action was not affected by nifedipine. ET-3 also stimulated a transient but significant release of LH from cells harvested from random cycle female rats, an effect that was not antagonized by a LHRH antagonist, but was blocked by nifedipine, suggesting the mobilization of extracellular calcium as a mechanism of action of ET-3. Nifedipine also reversed the acute stimulatory effect of ET-3 on GH secretion from these cells. Cerebroventricular injections of ET-3 (6 or 60 ng) failed to significantly alter PRL or LH secretion in conscious rats, suggesting that brain-derived ET does not act within the hypothalamus to alter the release of these two hormones. Similarly, iv infusion of even pressor doses of ET-3 (10, 30, or 300 ng) failed to significantly alter PRL, LH, or GH release; thus, it is unlikely that ET of peripheral origin acts within the gland. Our results suggest that locally produced ET may act as a neuroendocrine or paracrine factor controlling pituitary function in the rat.
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Hypothalamic endothelin: presence and effects related to fluid and electrolyte homeostasis.
Endothelin-3-like immunoreactivity (ET-3-ir) was detected in extracts of rat hypothalamic median eminence, and in the anterior and neurointermediate lobes of the pituitary at levels (ng ET-3/mg protein) exceeding those present in extracts of abdominal aorta. This ET-3-ir appeared authentic because radioimmunoassay (RIA) dose-response curves parallel to those of synthetic ET-3 could be constructed and this ET-3-ir comigrated on C-8 high-pressure liquid chromatography (HPLC) with synthetic ET-3. Endothelin-1-like immunoreactivity, on the other hand, was abundant in extracts of abdominal aorta and cerebral cortex and only minimally present in hypothalamus and anterior pituitary gland. Central administration of 11 and 23 pmol ET-3 resulted in significant (4.2- and 5.7-fold, respectively) elevations of plasma levels of vasopressin. Oxytocin levels were transiently, yet significantly, elevated (1.8-fold) by the higher dose of ET-3. These results, and our findings that central administration of ET-3 inhibits stimulated water drinking, suggest a physiologically important role for endogenously produced endothelin in the central mechanisms regulating fluid and electrolyte homeostasis.
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Physiologically important role for central oxytocin in the preovulatory release of luteinizing hormone.
Recently, our laboratory has provided evidence for a physiologically relevant stimulatory influence of oxytocin (OXY) on the preovulatory luteinizing hormone (LH) surge in cycling female rats. The present study evaluated whether this stimulatory effect of OXY on LH release is exerted at a central or peripheral site of action by comparing the ability of peripheral (intravenous) or central (intracerebroventricular) administration of OXY antisera to influence the preovulatory LH surge. The peripheral injection of a very large dose of OXY antisera (0.8 ml) caused a slight attenuation of the early stages of the LH surge. In contrast, the central administration of 5 microliters of OXY antisera completely abolished the preovulatory LH surge. The data support the hypothesis that OXY exerts a physiologically important stimulatory influence on the preovulatory LH surge which is mediated primarily at a central site of action.
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Pituitary site of action of endothelin: selective inhibition of prolactin release in vitro.
Endothelin-3 (ET-3) inhibited in a dose-dependent, significant fashion prolactin release from cultured anterior pituitary cells (ovariectomized female and intact male rat donors, ED50 = 5 X 10(-9) M). ET-3 in log doses ranging from 10(-11) to 10(-6) M did not alter significantly the release of luteinizing hormone, growth hormone or thyroid stimulating hormone. The inhibitory effect of ET-3 (rat, human) was specific for that molecule since ET-1 (porcine, human) was ineffective and was not due to an action on the dopamine receptor since the inhibitory action was still expressed in the presence of 100 nM domperidone. These data further suggest a role for neuropeptides of the posterior lobe in the control of lactotroph function.
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Cardiac hormones and neuroendocrine function.
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Animals↗
Interactions of dopaminergic and peptidergic factors in the control of prolactin release.
Oxytocin (OT) has been shown to play a role in the control of physiological PRL release and has been demonstrated to have a direct effect on the pituitary to stimulate PRL secretion. Administration of OT into the third ventricle, however, lowers PRL levels. This reduction could be mediated by either an inhibition of the release of endogenous OT into the hypohysial portal circulation or via an alteration in the release of some other PRL releasing (PRF) or PRL release-inhibiting (PIF) factor. In order to determine if centrally administered OT lowers PRL levels by increasing secretion of dopamine (DA) into the portal circulation, endogenous dopaminergic tone was blocked by injection of the DA antagonist domperidone (DOM). Subcutaneous administration of DOM resulted in elevated PRL levels which could be further augmented by iv infusion of OT (at 0.01 or 0.1 microgram OT/kg.min) or partially, but significantly, reduced by pretreatment with anti-OT antiserum (0.75 ml) indicating that under conditions of DA blockade, OT (which has little PRF activity during conditions of normal dopaminergic tone) can stimulate PRL secretion by a direct pituitary action. Treatment with DOM did not prevent, however, the reduction in PRL levels produced by central administration of OT (2 micrograms). This suggests that the effect of OT to alter PRL secretion when administered into the third ventricle was not mediated via an increase in DA release into the portal circulation. Furthermore, central administration of the OT antagonist CAV-259 (1-deamino-2-D-Trp-4-Val-8-Orn-OT) after DOM treatment resulted in a significant increase in PRL secretion indicating that endogenous levels of OT within the hypothalamus inhibit PRL secretion through a nondopaminergic mechanism. This stimulatory effect of the OT antagonist was not blocked by pretreatment with anti-OT antiserum (iv) which had been demonstrated previously to reduce the PRL surges in lactating mothers and steroid-primed ovariectomized rats, as well as to block the increase in PRL secretion seen after central administration of vasoactive intestinal peptide (VIP). Thus the central effect of OT to alter PRL secretion was probably not due to a change in the release of OT into the portal circulation. Intravenous administration of a VIP antagonist (D-4-Cl-6-Phe-17-Leu-VIP, previously demonstrated to be capable of reducing the PRL surge seen in lactating mothers) into DOM-treated rats does not alter PRL levels but blocks the ability of central administration of the OT antagonist CAV-259 to increase PRL levels under these conditions.(ABSTRACT TRUNCATED AT 400 WORDS)
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A nonoxytocinergic prolactin releasing factor and a nondopaminergic prolactin inhibiting factor in bovine neurointermediate lobe extracts: in vitro and in vivo studies.
Several peptidergic PRL-releasing factors (PRFs) have been described; however, none have been proven to be of primary physiological importance in the control of hormone release. Similarly dopamine withdrawal alone cannot completely explain the profiles of PRL secretion observed under a variety of conditions. We describe here the isolation in semipurified form of both a PRF and a PRL-inhibiting factor (PIF) from bovine neurointermediate lobe (NIL) extracts. Acid extracts of bovine NILs stimulated, in a dose-related manner, PRL release from cultured anterior pituitary cells, even after immunoabsorption of endogenous oxytocin from the extract. PIF and PRF activities were semipurified from NIL extracts by Sephadex chromatography and detected by in vitro and in vitro bioassays. The PRF material could be separated from oxytocin by gel sieving and was active in the presence of dopamine in vitro unlike synthetic oxytocin and in cell preparations in which the oxytocin-responsive lactotrophs had been removed by selective cytotoxin cell targeting using an oxytocin-ricin A chain cytotoxic conjugate. The PRF material stimulated PRL secretion in a dose-dependent fashion in conscious male rats after iv injection. The PIF material comigrated on sizing gel chromatography with immunoreactive oxytocin and was active in vitro during dopamine blockade with domperidone and in vivo in the presence of endogenous dopaminergic tone. These data suggest that novel factors present in the NIL might exert physiologically relevant control over lactotroph function and add to the growing literature on the presence of a PRF in the NIL.
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Destruction of the dorsal anterior hypothalamic region suppresses pulsatile release of follicle stimulating hormone but not luteinizing hormone.
The region of the paraventricular nucleus-dorsal anterior hypothalamic area (PVN-DAHA) previously was implicated in the control of tonic FSH secretion. However, the role that this hypothalamic area plays in governing pulsatile FSH release is unknown. To examine this question, radiofrequency (RF) lesions were produced bilaterally in the PVN-DAHA of adult female rats which had been ovariectomized for 4 weeks. Control animals received sham lesions. After a recovery period of 1 week, all rats were fitted with jugular cannulae. The next day sequential blood samples were withdrawn from conscious, undisturbed rats at 10-min intervals for 3 h. Control animals displayed secretory peaks of FSH in plasma with a frequency of 4.0 +/- 0.44/3-hour period (or 1 peak/40-50 min). LH in plasma pulsed at a frequency of 5.8 +/- 0.49 peaks/3 h (or 1 peak/20-30 min). Both of these control values were in agreement with previous studies. RF lesion of the PVN-DAHA reduced FSH peak frequency to 1.2 +/- 0.37 peaks/3 h (p less than 0.001) and also significantly suppressed the mean peak height and trough values for FSH (p less than 0.001). In contrast, none of these parameters for pulsatile LH secretion was altered by the lesion. Immediately after the 3-hour sampling period, synthetic LHRH (50 ng/100 g BW) was infused intravenously into rats and blood samples withdrawn 10 and 40 min later to determine whether the responsiveness of the pituitary gland had changed as a result of the lesion.(ABSTRACT TRUNCATED AT 250 WORDS)
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Oxytocin mediates the hypothalamic action of vasoactive intestinal peptide to stimulate prolactin secretion.
The ability of centrally administered vasoactive intestinal peptide (VIP) to stimulate PRL secretion when injected intracerebroventricularly could be due to leakage to the pituitary, where it is known to exert direct PRL-releasing activity, or to a hypothalamic action on its own release or that of another possible PRL-releasing factor. When 3 micrograms VIP were injected into the third ventricle of conscious ovariectomized rats, a significant (P less than 0.005) and transient elevation of plasma oxytocin (OT) levels was observed. When OVX rats were injected iv with 1 ml anti-OT serum 30 min before the central administration of 3 micrograms VIP, the PRL surge seen after VIP injection in normal rabbit serum-treated controls was completely absent. The PRL surge seen after central VIP administration was not significantly altered by iv saline infusion (1 ml over 30 min) or by infusion of a VIP antagonist [D-4-Cl-Phe6,Leu17]VIP at a dose of 0.5 microgram/kg.min in 1 ml saline for 30 min before the VIP injection. This was not due to the inability of the VIP antagonist to block the PRL-releasing factor activity of VIP, since it significantly antagonized that action both in vitro and in vivo in the suckling stimulation paradigm. However, the PRL surge was completely absent in ovariectomized rats pretreated by iv infusion of an OT antagonist, [deamino Cys1,D-Trp2,Val4,Orn8]OT, at a similar dose. This recruitment of OT by VIP indicates that it may act at more than one locus within the hypothalamo-pituitary axis to insure the coordinated control of PRL secretion.
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