- Email: [email protected]
Vasoactive intestinal polypeptide stimulates nonovarian progesterone secretion in rabbits
Peptides, Vol. 6, pp. 205--210, 1985. ©Ankho InternationalInc. Printed in the U.S.A.
01%-9781/85 $3.00 + .00
Vasoactive Intestinal Polypeptide Stimulates Nonovarian Progesterone Secretion in Rabbits C. M. F R E D E R I C K S / S . H . A S H T O N , W . F . A N D E R S O N , R. S. M A T H U R , L . E. L U N D Q U I S T * A N D S. L A N D G R E B E
Departments o f Physiology and Obstetrics and Gynecology, Medical University of South Carolina Charleston, SC 29425 and *Department o f Biology, Lander College, Greenwood, SC 29646 R e c e i v e d 10 D e c e m b e r 1984 FREDERICKS, C. M., S. H. ASHTON, W. F. ANDERSON, R. S. MATHUR, L. E. LUNDQUIST AND S. LANDGREBE. Vasoactive intestinal polypeptide stimulates nonovarian progesterone secretion in rabbits. PEPTIDES 6(2) 205-210, 1985.--Recent experiments conducted in this laboratory have shown that intravenous infusions of vasoactive intestinal polypeptide (VIP) induce significant increases in plasma progesterone (P) in female rabbits. The purpose of this study was to determine the organ source of this P and to clarify the mechanisms by which it is induced. Intravenous infusions of VIP (37.5, 75, and 150 pmol/kg per min for 60 min) produced acute dose-dependent increases in plasma P in intact estrous rabbits. In ovariectomized (OVX) animals, VIP infusion (75 pmol/kg per min) produced a P increase of the same magnitude. In animals both OVX and adrenalectomized (ADX), this VIP effect was eliminated. The only significant change noted in luteotropic hormone (LH) or follicle stimulating hormone (FSH) was a decrease in FSH immediately following VIP infusion (150 pmol/kg). VIP infusion significantly increased plasma cortisol in intact and OVX animals, but not in OVX/ADX animals. It is concluded that VIP primarily stimulates the adrenal component of P secretion in the rabbit, via mechanisms independent of LH or FSH. Vasoactive intestinal polypeptide
VIP
Progesterone
W H E N vasoactive intestinal polypeptide (VIP) was first isolated from the porcine duodenum in 1970 by Said and Mutt [29], its most likely physiologic function was some role in the regulation of gastrointestinal tract activity. Since that time, this peptide has been shown to be distributed in a wide variety of tissues and to exert numerous pharmacologic effects. VIP is localized primarily within elements of the central and peripheral nervous systems and satisfies many of the requirements of a "neurotransmitter" [5, l l, 12, 16, 25]. It now seems likely that VIP is released in vivo from central and peripheral neural sites and plays a role in the regulation of a number of physiologic processes, including various aspects of reproductive function [25]. The presence of VIP in the hypothalamus, anterior pituitary, and hypophyseal portal blood has suggested, for example, that VIP might be involved in the regulation of anterior pituitary hormone secretion [30,31]. Exogenous VIP can stimulate the release of prolactin (PRL) in rats, monkeys, and man and is now considered a probable PRLreleasing factor in these species [2, 6, 14, 19, 22, 30, 31, 34]. VIP is also present in the female reproductive tract itself and exogenous VIP has been shown to increase uterine blood flow [4,21] and to inhibit uterine and oviductal motility [7, 9,
Steroidogenesis
Adrenal gland
10, 23, 25, 33]. These effects have suggested a possible role for VIP in the regulation of reproductive tract motility, gamete transport, and regional blood flow [7, 10, 33]. Observations made in women further indicate that VIP may play a role in the control of uterine motility in late pregnancy and during labor, and in the control of vaginal blood flow and secretion during sexual stimulation [24,26]. Experiments conducted in this laboratory suggest that VIP may also play a role in regulating the secretion of certain ovarian steroids [8]. Specifically, intravenous infusions of VIP have been shown to induce significant increases in plasma P in female rabbits. These acute increases are dosedependent and seem to be independent o f any significant changes in plasma levels of estradiol (Ez), testosterone (T), or PRL. These increases in P occur both from the low levels seen in estrus and from the markedly elevated levels seen preceding ovulation and during pregnancy. In an effort to further characterize the VIP effect upon P secretion and to gain insight into its physiological significance, experiments were designed to determine whether: (1) VIP exerts this effect via stimulation of L H or F S H secretion; (2) this effect reflects increases in both the ovarian and adrenal components of P secretion in the rabbit.
~Requests for reprints should be addressed to Dr. Christopher M. Fredericks, Department of Physiology, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425.
205
F R E D E R I C K S ET AL.
206 METHOD
Animals The animals used throughout these investigations were virgin adult female New Zealand white rabbits. All were housed individually, with an ambient temperature of 21°C and a regulated light cycle of 14L: 10D. All animals were held for at least 3 weeks prior to study and were fed commercial rabbit chow and water ad lib.
Analysis of Plasma Hormone Concentrations At prescribed intervals, peripheral plasma levels of LH, F S H , cortisol, or P were measured in control and treated animals. F o r this purpose, 3-5 ml blood samples were collected via indwelling marginal ear vein catheters in heparinized syringes and placed in tubes containing 10.5 mg EDTA (K3). These samples were then centrifuged for 20 min and the plasma collected and frozen for subsequent analysis. Progesterone radioimmunoassay (RIA). Plasma P concentrations were determined by RIA according to the method of Mathur et al. [17]. Progesterone was extracted with n-hexane, dried, and reconstituted in methanol for assay. The sheep-generated P antibody used in these assays exhibited less than 1% cross-reactivity with 17-OH P and less than 0.1% cross-reactivity with cortisol, estradiol, and estriol. All assays were conducted in duplicate. Interassay variation was <15% and intra-assay variation was <10%. The minimal detectable quantity of P was 20 ng/dl. Gonadotropin RIA. Plasma samples were analyzed for the content of L H and F S H by specific homologous double antibody RIA with kits for rabbit L H and F S H supplied by Drs. S. Raiti and A. Parlow of the National Hormone and Pituitary Program. Highly purified F S H ( N I A D D K - F S H AFP-538-C) and L H (NIADDK-LH-AFP-559-B) were used for both iodination and for standard curve preparation. Antibodies against rabbit F S H (AFP-2-7-1) and L H (AFP-8-1-28) were raised in guinea pigs and supplied with the kits. The second antibody against guinea pig gamma globulin was prepared in this laboratory. The minimal detectable quantity per ml of serum was 0.63 ng in both the L H and F S H assay; These assays have been detailed previously [ 18]. Cortisol RIA. Plasma cortisol levels were analyzed using an automated test system and RIA reagents purchased from Micromedic Systems, Inc., (Horsham, PA). Experimental samples, standards, and cortisol r~5I-histamine were incubated in assay tubes pretreated with cortisol antibodies produced in rabbits. The minimal detectable quantity of cortisol was 0.3 /~g/dl. Interassay variation was <10% and intra-assay variation <7%.
Treatment Groups Three groups of animals were infused intravenously with VIP (Sigma Chemical Co., St. Louis, MO) or vehicle (lactated ringers). All infusions were made in conscious animals via indwelling ear vein catheters, at a rate of 0.21 ml/min for 60 min. Blood samples for hormone assay were drawn from the contralateral ear immediately preceding, and at prescribed intervals during or after these infusions. 1. Intact. Forty adult does in estrus were infused with vehicle or VIP (37.5, 75, or 150 pmol/kg per min), with no single animal receiving more than one concentration of VIP. 2. Ovariectomized (OVX). In order to eliminate the ovaries as a source of P, 9 adult does were surgically ovariectomized prior to VIP (75 pmol/kg per min) and vehicle infu-
150 pmol VIP m
500'
-1-
400'
tb
300
m
200,
IOO. ~ o iJJ Ztz:O300 ]
75 prnol VIP t
~-"' 2001 i.~o IOO] n.- o" Q_
oo] if)
5Z5 pmol VIP N,5
,00] O"
VEHICLE
0"
0
0.5
1.0
1.5
N,14
2.0
6.0
HOURS FIG. 1. Effects of intravenous infusions of VIP (37.5, 75, and 150 pmo1/kg per min) or vehicle upon plasma concentrations of P in estrous rabbits. Vertical bars represent mean---SEM. *Indicates values significantly different (p<0.05) from preinfusion values. VIP was infused from Time 0 to Time 1.0 (hours).
sion. The reproductive tract was exposed by a midline abdominal incision, the ovarian vessels ligated, and both ovaries removed. All surgery was performed under ketamine---(150 mg IM; Ketaset ®, Bristol Laboratories, Syracuse, NY) xylazine (50 mg IM; Rompun ®, Bayvet, Shawnee, KA) anesthesia, with maximal asepsis. Postoperative care included the administration of 1 or 2 intramuscular doses of procaine penicillin G and dihydrostreptomycin (Combiotic ®, Pfizer Inc., New York, NY). All animals were allowed to recover for 20-30 days before infusions were made. 3. Ovariectomized/Adrenalectomized (OVX/ADX). In order to eliminate both the ovaries and adrenal glands as sources of P, a group of 9 animals were ovariectomized and adrenalectomized. All animals were surgically ovariectomized as described above, and allowed to recover for 2 to 3 weeks before adrenalectomy was performed. In 4 animals, the right and left adrenal glands were removed in separate surgical procedures 2 to 3 weeks apart. In the remaining 5 animals, both adrenal glands were removed during the same procedure. In all cases, the adrenal glands were exposed by a midline incision reaching from the xiphoid process to the umbilicus. The left adrenal could be seen through the posterior peritoneum in the angle formed by the inferior vena cava and the left renal vein and could be easily excised once its vascular stalk was ligated. The right adrenal was found closely adhered to the posterior wall of the vena cava, near the entrance of the right renal vein. This gland had to be carefully teased away from the wall of the vena cava around
VIP S T I M U L A T I O N O F A D R E N A L P R O G E S T E R O N E 150 pmol VIP
207
N-8
OVX/ADX
'°°1
~~.,_ 20.
O'
7,/
C.
VEHICLE i ,~
2.0
OVX
300"
0.5
150 pmol VIP N=8
z o ~
200-
o
:;-lu) --j
VEHICLE
ct. 0.5} ~. 0
0
N=6
&
0.5
1.0
1.5
2.0
6.0
HOURS
FIG. 2. Effects of intravenous infusion of VIP (150 pmol/kg per min) or vehicle upon plasma concentrations of LH and FSH in estrous rabbits. Vertical bars represent mean_+SEM. *Indicates values significantly different (p<0.05) from preinfusion values. VIP was infused from Time 0 to Time 1.0 (hours).
ta.l z 0 12: laJ
N,9
I00
co IxJ (D 0 0 n ,¢ 3 0 0 ' ~E CO ._1 n
a.
// ,
INTACT
200' N=9
I00' Infusion I
//
0
o its margin and the remaining attachment carefully ligated before it could be removed. All animals received 2.0 mg cortisone acetate (CA; Cottone acetate ®, Merck, Sharp and Dohme, Westpoint, PA) and 1.5 mg deoxycorticosterone (DOCA acetate®; Organon, Inc., West Orange, NJ) intramuscularly for 3 days prior to surgery. Following surgery, all animals received this dose for 5 days and then were maintained by 0.7 mg CA and 0.5 mg DOCA given every third day. The administration of both CA and DOCA was stopped at least 3 days prior to VIP or vehicle infusion and the acquisition of blood samples for hormone assay. Drinking water and NaCl (0.9%) solution were provided to all OVX/ADX animals postoperatively. All animals were allowed to recover for 2-3 weeks before any infusions were made. The VIP used throughout these studies was a synthetic porcine molecule.
Statistical Analysis Sequential F S H , LH, and P values within animal groups were tested by analysis of variance for repeated measures and, when significant, differences in means were compared by the Newman-Keuls test. Differences among cortisol values within animal groups were assessed by a paired t-test. The significance of differences between animal groups at any one time was determined by pooling the hormone values within each group and comparing these pools using the Student's t-test. Alpha levels o f p <0.05 were regarded as significant.
0.5
,o
,5
A.
el0
HOURS
FIG. 3. Effects of intravenous infusion of VIP (75 pmol/kg per min; solid circle) or vehicle (open circle) upon plasma concentrations of P in: (A) intact estrous rabbits, (B) ovariectomized rabbits, (C) ovariectomized and adrenalectomized rabbits. Vertical bars represent mean_+SEM. *Indicates values significantly different (p<0.05) from preinfusion values.
RESULTS In confirmation of results previously reported by this laboratory [8], VIP (37.5, 75, and 150 pmol/kg per min) infused intravenously for 60 min in estrous rabbits produced acute dose-dependent increases in plasma P (Fig. 1). An analogous infusion of vehicle (lactated ringers) produced no significant change in this hormone. In an effort to determine whether gonadotropins were involved in the effects of infused VIP upon P secretion, plasma concentrations of L H and F S H were determined in association with VIP (150 pmol/kg per min) or vehicle infusion (Fig. 2). The only significant change which was noted was a decrease in the F S H concentration of the plasma drawn immediately following the VIP infusion. In order to determine the organ source of the VIPstimulated P secretion, animals were OVX or OVX/ADX and infused with VIP (75 pmol/kg per min) or vehicle (Fig. 3). In nine animals in which both ovaries were removed, VIP infusion produced an increase in P which was of the same
208
FREDERICKS ET AL.
magnitude as that produced in intact animals. In nine animals in which both adrenal glands were also removed, the VIP effect upon plasma P was eliminated. OXY/ADX but not OVX significantly lowered the preinfusion levels of P. In all three animal groups, plasma cortisol levels were also determined. A significant increase in plasma cortisol was observed in association with VIP (75 pmol/kg/min) infusion in both intact and OVX animals, but not in OVX/ADX animals. Although plasma cortisol (4.3_+0.5 ~g/dl) remained in OVX/ADX animals, it was significantly (p<0.001) less than pre-operative levels (9.3_+ 1.0/zg/dl). All the OVX and OVX/ADX animals reported herein healed well, with uneventful postoperative courses. In the OVX/ADX group there were no apparent differences in the course of recovery or in the response to infusion between those ADX in two procedures and those in a single procedure. In all cases a thorough autopsy was performed at the end of experimentation. Particular care was taken to determine whether any ovarian or adrenal tissue remained in these animals. In no OVX or OVX/ADX animals was any ovarian tissue found. In 6 of the 9 0 V X / A D X animals, nodes (1-2 mm diam.) of suspected adrenal tissue were found attached to the inferior vena cava near the excision site. Histological examination confirmed that these nodes were composed largely of adrenal cortical cells. With an average approximate diameter of 1.5 mm, each node represented a calculated <2-3% of the volume of an average whole adrenal gland (5-6 mm diam.). No positive correlation was detected between the amount of accessory adrenal tissue noted at autopsy and the post-OVX/ADX plasma levels of cortisol. DISCUSSION
During the last several years tremendous growth has occurred in the attention focused on the biological substance, VIP. This polypeptide, which was once of interest primarily as a putative gut hormone, is now known to be present in the neural elements of many tissues and to exert a broad spectrum of pharmacological effects. Despite this attention, however, relatively little is known about the actual physiologic significance of this substance. One area in which the biologic importance of VIP is beginning to be defined is reproduction. Considerable evidence suggests that VIP may play a role in both central and peripheral reproductive processes. A number of investigators have concluded, for example, that VIP is a probable PRLreleasing factor in rats, monkeys, and man [2, 6, 14, 19, 22, 30-32, 34]. Others have suggested that VIP may function as a neurotransmitter in the female genital tract itself, and serve to influence the contractility of the muscular elements of the vasculature, as well as the ovarian follicle, the oviduct and the uterus [4, 7, 9, 10, 21, 23, 25, 33]. The observation reported by this laboratory that VIP infused into rabbits markedly elevated plasma levels of P, indicated that VIP might also play a role in the regulation of ovarian steroid secretion. The data summarized in this report, as well as that reported previously by Fredericks et al. [8], suggest, however, that this effect upon P is not related to reproductive processes per se. Any direct involvement of VIP in the central regulation of P secretion, particularly as a component of reproductive function, would most likely involve VIP effects upon the anterior pituitary hormones, LH, F S H , and PRL. There is little evidence that this is occurring in the rabbit.
TABLE 1 PLASMA CORTISOL (p,g/dl)*
Time (hr)
75 pmol VIP
Vehicle
Intact 0
10.5 ± 1.3 (8)
1
19.2 ± 1.5 (8)**
12.0 ± 0.6 (6) 10.4 -+ 1.2 (6)
Ovariectomized (OVX) 0 1
13.0 ± 0.4 (9) 21.8 ± 2.1 (9)**
Not Done
Ovariectomized/Adrenalectomized (OVX/ADX) 4.3 ± 0.5 (9) 6.6 ± 1.0 (9)
Not Done
*Values are mean - S.E.M.; number of animals in parentheses. VIP (75 pmoi/kg per min) or vehicle (lactated ringers) was infused intravenously for 1 hr (T+0 to T+ 1). **Indicates values significantly different (p<0.01) from corresponding preinfusion values. OVX/ADX pre- and post-infusion values are significantly different (p<0.001) from corresponding values of both intact and OVX.
Progesterone-stimulating infusions of VIP are not associated with increased secretion of F S H or LH, in fact a slight inhibition of F S H was noted. Also, if the P increases in the rabbit were the result of a significant increase in LH, ovulation and follicular luteinization would have been expected. This did not occur. Other investigators have shown similarly that infused VIP does not increase LH or F S H in rats or humans [22,34]. By the same token, Fredericks et al. [8] have previously shown that the effect of VIP upon P in the rabbit is not associated with any significant change in plasma PRL levels, nor is it affected by a PRL-lowering treatment with bromocriptine. Not only are the gonadotropins apparently not involved, but the ovaries themselves do not appear to be the source of the VIP-stimulated P. The observation that OVX has little effect upon the P response, while it is abolished by ADX, suggests that the source of the P is, in fact, the adrenal gland. Although substantial amounts of P are synthesized by both the adrenals and ovaries in the rabbit [27], VIP appears to stimulate primarily the adrenal component. The effect of VIP upon adrenal secretion is also indicated by the observation that P only increased when cortisol significantly increased, such as in the intact and OVX animals. In the OVX/ADX animals, in which no significant increase in cortisol accompanied the VIP infusion, P did not increase. The mechanism of this VIP effect, however, remains unknown. Any central effect upon the adrenal cortex would be mediated by increased pituitary ACTH release. To date, however, it is not at all clear whether VIP has any significant effects upon ACTH release, either in vivo or in vitro. Experiments conducted in intact animals have been contradictory. Bataille et al. [1] infused VIP into normal humans and observed a significant increase in serum ACTH. Ottesen et al. [22], on the other hand, in similar experiments found no increase in plasma cortisol. When infused into normal rats intracisternally, V1P increased ACTH release, but not when
VIP S T I M U L A T I O N O F A D R E N A L P R O G E S T E R O N E infused intravenously [13]. VIP has been shown to produce a dose-dependent stimulation of A C T H release from a line of human pituitary tumor cells [20], but no significant change in the A C T H released from purified rat pituitary cells [28] or incubated rat pituitary tissue [13]. VIP produces a stimulation of steroidogenesis in cultured mouse adrenal cortical tumor cells as great as that produced by A C T H [15]. These effects, however, require high VIP doses and appear to be exerted through receptors distinct from those of A C T H [3]. VIPergic nerve fibers are present in the adrenal medulla and cortex of the rat [12] and could conceivably exert a local effect upon P secretion. It remains to be determined whether the VIP effects upon P secretion reflect a direct effect upon the pituitary-adrenal axis, and hence may suggest some physiological role for VIP in the regulation of adrenal secretion, or whether this increase in P merely accompanies a generalized activation of adrenal mechanisms resulting from VIP effects upon other biologic processes. Interestingly, a significant amount o f P remained in the plasma of OVX/ADX animals. The source of this P, however, has yet to be determined. It is unlikely that it is se-
209
creted by the adrenal tissue remaining postoperatively in some animals, since the amount of this tissue was small (calculated to be less than 2-3% of an intact gland) and was shown to be unable to support the VIP effect upon either P or cortisol. In addition, no positive correlation could be established between the amount of adrenal tissue noted at autopsy and the plasma levels of P. It is more likely that the P is coming from some other tissue, perhaps from the uterus. Overstrom and Black (1980; personal communication, 1984) have shown that plasma P remains after OVX/ADX in rabbits and that rabbit endometrial cells can synthesize P in culture. This suggests that in vivo the rabbit uterus may contribute to overall levels of circulating P.
ACKNOWLEDGEMENTS The authors would like to thank Dr. Thomas Mills, Medical College of Georgia, for his generous support of this project in contributing the LH and FSH assays. We also thank Judy Kameoka for her diligent technical assistance. This work was supported in part by the South Carolina State Appropriation for Biomedical Research.
REFERENCES I. Bataille, D., J. N. Talbot, G. Miihaud, V. Mutt and G. Rosselin. Effet du peptide intestinal vasoactif (VIP) sur la secretion de prolactine chez l'homme. CR Acad Sci (Paris) 292: 311-313, 1981. 2. Besson, J., W. H. Rotsztein and D. Bataille. Involvement of VIP in neuroendocrine functions. In: Vasoactive Intestinal Polypeptide, edited by Sami I. Said. New York: Raven Press, 1982, pp. 253-262. 3. Birnbaum, R. S., M. Alfonzo and J. Kowal. Vasoactive intestinal peptide- and adrenocorticotropin-stimulated adenylcyclase in cultured adrenal tumor cells: Evidence for a specific vasoactive intestinal polypeptide receptor. Endocrinology 1116: 12701275, 1980. 4. Clark, K. E., E. G. Mills, S. J. Stys and A. E. Seeds. Effects of vasoactive polypeptides on the uterine vasculature. Am J Obstet Gynecol 139: 182-188, 1981. 5, Fahrenkrug, J. VIP as a neurotransmitter in the peripheral nervous system. In: Vasoactive Intestinal Polypeptide, edited by Sami I. Said. New York: Raven Press, 1982, pp. 361-372. 6. Frawley, L. S. and J. D. Neill. Stimulation of prolactin secretion in Rhesus monkeys by vasoactive intestinal polypeptide. Neuroendocrinology 33: 7%83, 1981. 7. Fredericks, C. M. and S. H. Ashton. Effect of vasoactive intestinal polypeptide (VIP) on the in vitro and in vivo motility of the rabbit reproductive tract. Fertil Steril 37: 845-850, 1982. 8. Fredericks, C. M., L. E. Lundquist, R. S. Mathur, S. H. Ashton and S. C. Landgrebe. Effects of vasoactive intestinal polypeptide upon ovarian steroids, ovum transport, and fertility in the rabbit. Biol Reprod 28: 1052-1060, 1983. 9. Helm, G., B. Ottesen, J. Fahrenkrug, J. J. Larsen, C. Owman, N. O. Sjoberg, B. Stolberg, F. Sundler and B. Walles. Vasoactive intestinal polypeptide (VIP) in the human reproductive tract: Distribution and motor effects. Biol Reprod 25: 227-234, 1981. 10. Helm, G., S. Hakanson, S. Leander, C. Owman, N. O. Sjoberg and B. Sporrong. Neurogenic relaxation mediated by vasoactive intestinal polypeptide (VIP) in the isthmus of the human fallopian tube. Regal Pept 3: 145-153, 1982. II. Hokanson, R., F. Sundler and R. Uddman. Distribution and topography of peripheral VIP nerve fibers: Functional implications. In: Vasoactive Intestinal Polypeptide, edited by Sami I. Said. New York: Raven Press, 1982, pp. 121-144.
12. Hokfelt, T., M. Schultzberg, J. M. LundbeI'g, K. Fuxe, V. Mutt, J. Fahrenkrug and S. I. Said. Distribution of vasoactive intestinal polypeptide in the central and peripheral nervous systems as revealed by immunocytochemistry. In: Vasoactive Intestinal Polypeptide, edited by Sami I. Said. New York: Raven Press, 1982, pp. 65-90. 13. Itoh, S., R. Hirota and G. Katsuura. Effect of cholecystokinin octapeptide and vasoactive intestinal polypeptide on adrenocortical secretion in the rat. Jpn J Physiol 32: 553-560, 1982. 14. Kato, Y., Y. Iwasaki, J. Iwasaki, H. Abe, N. Yanaihara and H. Imura. Prolactin release by vasoactive intestinal polypeptide in rats. Endocrinology 103: 554-558, 1978. 15. Kowal, J. VIP effects on adrenocortical cell functions. In: Vasoactive Intestinal Polypeptide, edited by Sami I. Said. New York: Raven Press, 1982, pp. 277-284. 16. Marley, P. and P. Emson. VIP as a neurotransmitter in the central nervous system. In: Vasoactive Intestinal Polypeptide, edited by Sami I. Said. New York: Raven Press, 1982, pp. 341360. 17. Mathur, R. S., S. Landgrebe and H. O. Williamson. Progesterone, 17-hydroxyprogesterone, estradiol, and estriol in late pregnancy and labor. Am J Obstet Gynecol 136: 25-30, 1980. 18. Mills, T. M. and R. J. Gerardot. Dissociation of copulation from ovulation in pregnant rabbits. Biol Reprod 30: 1243-1252, 1984. 19. Nicosia, S., A. Spada and G. Giannattasio. Effects of vasoactive intestinal polypeptide on the pituitary gland. In: Vasoactive Intestinal Polypeptide, edited by Sami I. Said. New York: Raven Press, 1982, pp. 263-275. 20. Oliva, D., S. Nicosia, A. Spada and G. Giannattasio. VIP stimulates ACTH release and adenylate cyclase in human ACTHsecreting pituitary adenomas. Eur J Pharmacol 83: 101-105, 1982. 21. Ottesen, B. and J. Fahrenkrug. Effect of vasoactive intestinal polypeptide (VIP) upon myometrial blood flow in non-pregnant rabbit. Acta Physiol Scand 112: 195-201, 1981. 22. Ottesen, B., A. N. Anderson, T. Gerstenberg, H. Ulrichsen, T. Manthorpe and J. Fahrenkrug. VIP stimulates prolactin release in women. Lancet 2: 696, 1981.
210 23. Ottesen, B., J. J. Larsen, J. Fahrenkrug, M. Stjernquist and F. Sundler. Distribution and motor effects of VIP in female genital tract, Am J Physiol 240: E32-36, 1981. 24. Ottesen, B., H. Ulrichsen, J. Fahrenkrug, J. J. Larsen, G. Wagner, L. Schierup and F. Sondergaard. Vasoactive intestinal polypeptide and the female genital tract: Relationship to reproductive phase and delivery. Am J Obstet Gynecol 143: 414-420, 1982. 25. Ottesen, B., F. Sondergaard and J. Fahrenkrug. Neuropeptides in the regulation of female genital smooth muscle contractility. Acta Obstet Gynecol Scand 67: 591-592, 1983. 26. Ottesen, B., T. Gerstenberg, H. Ulrichsen, T. Manthorpe, J. Fahrenkrug and G. Wagner. Vasoactive intestinal polypeptide (VIP) increases vaginal blood flow and inhibits uterine smooth muscle activity in women. Eur J Clin Invest 13: 321-324, 1983. 27. Overstrom, E. W. and D. L. Black. Effect of ovariectomy and adrenalectomy on peripheral plasma concentrations of estradiol and progesterone in the rabbit. Biol Reprod 22: Suppl, 134A (Abstract), 1980. 28. Rotsztejn, W. H., L. Benoist, J. Besson, G. Beraud, M. T. Bluet-Pajot, C. Kordon, G. Rosselin and J. Duval. Effect of vasoactive intestinal polypeptide (VIP) on the release of adenohypophyseal hormones from purified cells obtained by unit gravity sedimentation: Inhibition by dexamethasone of VIP-induced prolactin release. Neuroendocrinology 31: 282286, 1980.
FREDERICKS ET AL. 29. Said, S. I. and V. Mutt. Polypeptide with broad biological activity: Isolation from small intestine. Science 160: 1217-1218, 1970. 30. Said, S. I. and J. C. Porter. Vasoactive intestinal polypeptide release into hypophyseal portal blood. Life Sci 24: 227-230, 1979. 31. Shumatsu, A., Y. Kato, N. Matsushita, H. Katakami, B. Yanalhara and H. Imura. Immunoreactive vasoactive intestinal polypeptide in rat hypophyseal portal blood. Endocrinology 108: 395-398, 1981. 32. Shumatsu, A., Y. Kato, H. Ohta, K. Tojo, Y. Kabayama, T. Inoue, N. Yanaihara and H. Imura. Involvement of hypothalamic vasoactive intestinal polypeptide (VIP) in prolactin secretion induced by serotonin in rats. Proc Exp Biol M5: 414-416, 1984. 33. Strom, C., J. M. Lundberg, H. Ahlaman, A. Dahlstrom, J. Fahrenkrug and T. Hokfelt. On the VIPergic innervation of the uterotubal junction. Acta Physiol Scand I l l : 213-215, 1981. 34. Vijayan, E., W. K. Samson, S. I. Said and S. M. McCann. Vasoactive intestinal polypeptide: Evidence for a hypothalamic site of action to release growth hormone, luteinizing hormone, and prolactin in conscious ovariectomized rats. Endocrinology 104: 53-57, 1979.
01%-9781/85 $3.00 + .00
Vasoactive Intestinal Polypeptide Stimulates Nonovarian Progesterone Secretion in Rabbits C. M. F R E D E R I C K S / S . H . A S H T O N , W . F . A N D E R S O N , R. S. M A T H U R , L . E. L U N D Q U I S T * A N D S. L A N D G R E B E
Departments o f Physiology and Obstetrics and Gynecology, Medical University of South Carolina Charleston, SC 29425 and *Department o f Biology, Lander College, Greenwood, SC 29646 R e c e i v e d 10 D e c e m b e r 1984 FREDERICKS, C. M., S. H. ASHTON, W. F. ANDERSON, R. S. MATHUR, L. E. LUNDQUIST AND S. LANDGREBE. Vasoactive intestinal polypeptide stimulates nonovarian progesterone secretion in rabbits. PEPTIDES 6(2) 205-210, 1985.--Recent experiments conducted in this laboratory have shown that intravenous infusions of vasoactive intestinal polypeptide (VIP) induce significant increases in plasma progesterone (P) in female rabbits. The purpose of this study was to determine the organ source of this P and to clarify the mechanisms by which it is induced. Intravenous infusions of VIP (37.5, 75, and 150 pmol/kg per min for 60 min) produced acute dose-dependent increases in plasma P in intact estrous rabbits. In ovariectomized (OVX) animals, VIP infusion (75 pmol/kg per min) produced a P increase of the same magnitude. In animals both OVX and adrenalectomized (ADX), this VIP effect was eliminated. The only significant change noted in luteotropic hormone (LH) or follicle stimulating hormone (FSH) was a decrease in FSH immediately following VIP infusion (150 pmol/kg). VIP infusion significantly increased plasma cortisol in intact and OVX animals, but not in OVX/ADX animals. It is concluded that VIP primarily stimulates the adrenal component of P secretion in the rabbit, via mechanisms independent of LH or FSH. Vasoactive intestinal polypeptide
VIP
Progesterone
W H E N vasoactive intestinal polypeptide (VIP) was first isolated from the porcine duodenum in 1970 by Said and Mutt [29], its most likely physiologic function was some role in the regulation of gastrointestinal tract activity. Since that time, this peptide has been shown to be distributed in a wide variety of tissues and to exert numerous pharmacologic effects. VIP is localized primarily within elements of the central and peripheral nervous systems and satisfies many of the requirements of a "neurotransmitter" [5, l l, 12, 16, 25]. It now seems likely that VIP is released in vivo from central and peripheral neural sites and plays a role in the regulation of a number of physiologic processes, including various aspects of reproductive function [25]. The presence of VIP in the hypothalamus, anterior pituitary, and hypophyseal portal blood has suggested, for example, that VIP might be involved in the regulation of anterior pituitary hormone secretion [30,31]. Exogenous VIP can stimulate the release of prolactin (PRL) in rats, monkeys, and man and is now considered a probable PRLreleasing factor in these species [2, 6, 14, 19, 22, 30, 31, 34]. VIP is also present in the female reproductive tract itself and exogenous VIP has been shown to increase uterine blood flow [4,21] and to inhibit uterine and oviductal motility [7, 9,
Steroidogenesis
Adrenal gland
10, 23, 25, 33]. These effects have suggested a possible role for VIP in the regulation of reproductive tract motility, gamete transport, and regional blood flow [7, 10, 33]. Observations made in women further indicate that VIP may play a role in the control of uterine motility in late pregnancy and during labor, and in the control of vaginal blood flow and secretion during sexual stimulation [24,26]. Experiments conducted in this laboratory suggest that VIP may also play a role in regulating the secretion of certain ovarian steroids [8]. Specifically, intravenous infusions of VIP have been shown to induce significant increases in plasma P in female rabbits. These acute increases are dosedependent and seem to be independent o f any significant changes in plasma levels of estradiol (Ez), testosterone (T), or PRL. These increases in P occur both from the low levels seen in estrus and from the markedly elevated levels seen preceding ovulation and during pregnancy. In an effort to further characterize the VIP effect upon P secretion and to gain insight into its physiological significance, experiments were designed to determine whether: (1) VIP exerts this effect via stimulation of L H or F S H secretion; (2) this effect reflects increases in both the ovarian and adrenal components of P secretion in the rabbit.
~Requests for reprints should be addressed to Dr. Christopher M. Fredericks, Department of Physiology, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425.
205
F R E D E R I C K S ET AL.
206 METHOD
Animals The animals used throughout these investigations were virgin adult female New Zealand white rabbits. All were housed individually, with an ambient temperature of 21°C and a regulated light cycle of 14L: 10D. All animals were held for at least 3 weeks prior to study and were fed commercial rabbit chow and water ad lib.
Analysis of Plasma Hormone Concentrations At prescribed intervals, peripheral plasma levels of LH, F S H , cortisol, or P were measured in control and treated animals. F o r this purpose, 3-5 ml blood samples were collected via indwelling marginal ear vein catheters in heparinized syringes and placed in tubes containing 10.5 mg EDTA (K3). These samples were then centrifuged for 20 min and the plasma collected and frozen for subsequent analysis. Progesterone radioimmunoassay (RIA). Plasma P concentrations were determined by RIA according to the method of Mathur et al. [17]. Progesterone was extracted with n-hexane, dried, and reconstituted in methanol for assay. The sheep-generated P antibody used in these assays exhibited less than 1% cross-reactivity with 17-OH P and less than 0.1% cross-reactivity with cortisol, estradiol, and estriol. All assays were conducted in duplicate. Interassay variation was <15% and intra-assay variation was <10%. The minimal detectable quantity of P was 20 ng/dl. Gonadotropin RIA. Plasma samples were analyzed for the content of L H and F S H by specific homologous double antibody RIA with kits for rabbit L H and F S H supplied by Drs. S. Raiti and A. Parlow of the National Hormone and Pituitary Program. Highly purified F S H ( N I A D D K - F S H AFP-538-C) and L H (NIADDK-LH-AFP-559-B) were used for both iodination and for standard curve preparation. Antibodies against rabbit F S H (AFP-2-7-1) and L H (AFP-8-1-28) were raised in guinea pigs and supplied with the kits. The second antibody against guinea pig gamma globulin was prepared in this laboratory. The minimal detectable quantity per ml of serum was 0.63 ng in both the L H and F S H assay; These assays have been detailed previously [ 18]. Cortisol RIA. Plasma cortisol levels were analyzed using an automated test system and RIA reagents purchased from Micromedic Systems, Inc., (Horsham, PA). Experimental samples, standards, and cortisol r~5I-histamine were incubated in assay tubes pretreated with cortisol antibodies produced in rabbits. The minimal detectable quantity of cortisol was 0.3 /~g/dl. Interassay variation was <10% and intra-assay variation <7%.
Treatment Groups Three groups of animals were infused intravenously with VIP (Sigma Chemical Co., St. Louis, MO) or vehicle (lactated ringers). All infusions were made in conscious animals via indwelling ear vein catheters, at a rate of 0.21 ml/min for 60 min. Blood samples for hormone assay were drawn from the contralateral ear immediately preceding, and at prescribed intervals during or after these infusions. 1. Intact. Forty adult does in estrus were infused with vehicle or VIP (37.5, 75, or 150 pmol/kg per min), with no single animal receiving more than one concentration of VIP. 2. Ovariectomized (OVX). In order to eliminate the ovaries as a source of P, 9 adult does were surgically ovariectomized prior to VIP (75 pmol/kg per min) and vehicle infu-
150 pmol VIP m
500'
-1-
400'
tb
300
m
200,
IOO. ~ o iJJ Ztz:O300 ]
75 prnol VIP t
~-"' 2001 i.~o IOO] n.- o" Q_
oo] if)
5Z5 pmol VIP N,5
,00] O"
VEHICLE
0"
0
0.5
1.0
1.5
N,14
2.0
6.0
HOURS FIG. 1. Effects of intravenous infusions of VIP (37.5, 75, and 150 pmo1/kg per min) or vehicle upon plasma concentrations of P in estrous rabbits. Vertical bars represent mean---SEM. *Indicates values significantly different (p<0.05) from preinfusion values. VIP was infused from Time 0 to Time 1.0 (hours).
sion. The reproductive tract was exposed by a midline abdominal incision, the ovarian vessels ligated, and both ovaries removed. All surgery was performed under ketamine---(150 mg IM; Ketaset ®, Bristol Laboratories, Syracuse, NY) xylazine (50 mg IM; Rompun ®, Bayvet, Shawnee, KA) anesthesia, with maximal asepsis. Postoperative care included the administration of 1 or 2 intramuscular doses of procaine penicillin G and dihydrostreptomycin (Combiotic ®, Pfizer Inc., New York, NY). All animals were allowed to recover for 20-30 days before infusions were made. 3. Ovariectomized/Adrenalectomized (OVX/ADX). In order to eliminate both the ovaries and adrenal glands as sources of P, a group of 9 animals were ovariectomized and adrenalectomized. All animals were surgically ovariectomized as described above, and allowed to recover for 2 to 3 weeks before adrenalectomy was performed. In 4 animals, the right and left adrenal glands were removed in separate surgical procedures 2 to 3 weeks apart. In the remaining 5 animals, both adrenal glands were removed during the same procedure. In all cases, the adrenal glands were exposed by a midline incision reaching from the xiphoid process to the umbilicus. The left adrenal could be seen through the posterior peritoneum in the angle formed by the inferior vena cava and the left renal vein and could be easily excised once its vascular stalk was ligated. The right adrenal was found closely adhered to the posterior wall of the vena cava, near the entrance of the right renal vein. This gland had to be carefully teased away from the wall of the vena cava around
VIP S T I M U L A T I O N O F A D R E N A L P R O G E S T E R O N E 150 pmol VIP
207
N-8
OVX/ADX
'°°1
~~.,_ 20.
O'
7,/
C.
VEHICLE i ,~
2.0
OVX
300"
0.5
150 pmol VIP N=8
z o ~
200-
o
:;-lu) --j
VEHICLE
ct. 0.5} ~. 0
0
N=6
&
0.5
1.0
1.5
2.0
6.0
HOURS
FIG. 2. Effects of intravenous infusion of VIP (150 pmol/kg per min) or vehicle upon plasma concentrations of LH and FSH in estrous rabbits. Vertical bars represent mean_+SEM. *Indicates values significantly different (p<0.05) from preinfusion values. VIP was infused from Time 0 to Time 1.0 (hours).
ta.l z 0 12: laJ
N,9
I00
co IxJ (D 0 0 n ,¢ 3 0 0 ' ~E CO ._1 n
a.
// ,
INTACT
200' N=9
I00' Infusion I
//
0
o its margin and the remaining attachment carefully ligated before it could be removed. All animals received 2.0 mg cortisone acetate (CA; Cottone acetate ®, Merck, Sharp and Dohme, Westpoint, PA) and 1.5 mg deoxycorticosterone (DOCA acetate®; Organon, Inc., West Orange, NJ) intramuscularly for 3 days prior to surgery. Following surgery, all animals received this dose for 5 days and then were maintained by 0.7 mg CA and 0.5 mg DOCA given every third day. The administration of both CA and DOCA was stopped at least 3 days prior to VIP or vehicle infusion and the acquisition of blood samples for hormone assay. Drinking water and NaCl (0.9%) solution were provided to all OVX/ADX animals postoperatively. All animals were allowed to recover for 2-3 weeks before any infusions were made. The VIP used throughout these studies was a synthetic porcine molecule.
Statistical Analysis Sequential F S H , LH, and P values within animal groups were tested by analysis of variance for repeated measures and, when significant, differences in means were compared by the Newman-Keuls test. Differences among cortisol values within animal groups were assessed by a paired t-test. The significance of differences between animal groups at any one time was determined by pooling the hormone values within each group and comparing these pools using the Student's t-test. Alpha levels o f p <0.05 were regarded as significant.
0.5
,o
,5
A.
el0
HOURS
FIG. 3. Effects of intravenous infusion of VIP (75 pmol/kg per min; solid circle) or vehicle (open circle) upon plasma concentrations of P in: (A) intact estrous rabbits, (B) ovariectomized rabbits, (C) ovariectomized and adrenalectomized rabbits. Vertical bars represent mean_+SEM. *Indicates values significantly different (p<0.05) from preinfusion values.
RESULTS In confirmation of results previously reported by this laboratory [8], VIP (37.5, 75, and 150 pmol/kg per min) infused intravenously for 60 min in estrous rabbits produced acute dose-dependent increases in plasma P (Fig. 1). An analogous infusion of vehicle (lactated ringers) produced no significant change in this hormone. In an effort to determine whether gonadotropins were involved in the effects of infused VIP upon P secretion, plasma concentrations of L H and F S H were determined in association with VIP (150 pmol/kg per min) or vehicle infusion (Fig. 2). The only significant change which was noted was a decrease in the F S H concentration of the plasma drawn immediately following the VIP infusion. In order to determine the organ source of the VIPstimulated P secretion, animals were OVX or OVX/ADX and infused with VIP (75 pmol/kg per min) or vehicle (Fig. 3). In nine animals in which both ovaries were removed, VIP infusion produced an increase in P which was of the same
208
FREDERICKS ET AL.
magnitude as that produced in intact animals. In nine animals in which both adrenal glands were also removed, the VIP effect upon plasma P was eliminated. OXY/ADX but not OVX significantly lowered the preinfusion levels of P. In all three animal groups, plasma cortisol levels were also determined. A significant increase in plasma cortisol was observed in association with VIP (75 pmol/kg/min) infusion in both intact and OVX animals, but not in OVX/ADX animals. Although plasma cortisol (4.3_+0.5 ~g/dl) remained in OVX/ADX animals, it was significantly (p<0.001) less than pre-operative levels (9.3_+ 1.0/zg/dl). All the OVX and OVX/ADX animals reported herein healed well, with uneventful postoperative courses. In the OVX/ADX group there were no apparent differences in the course of recovery or in the response to infusion between those ADX in two procedures and those in a single procedure. In all cases a thorough autopsy was performed at the end of experimentation. Particular care was taken to determine whether any ovarian or adrenal tissue remained in these animals. In no OVX or OVX/ADX animals was any ovarian tissue found. In 6 of the 9 0 V X / A D X animals, nodes (1-2 mm diam.) of suspected adrenal tissue were found attached to the inferior vena cava near the excision site. Histological examination confirmed that these nodes were composed largely of adrenal cortical cells. With an average approximate diameter of 1.5 mm, each node represented a calculated <2-3% of the volume of an average whole adrenal gland (5-6 mm diam.). No positive correlation was detected between the amount of accessory adrenal tissue noted at autopsy and the post-OVX/ADX plasma levels of cortisol. DISCUSSION
During the last several years tremendous growth has occurred in the attention focused on the biological substance, VIP. This polypeptide, which was once of interest primarily as a putative gut hormone, is now known to be present in the neural elements of many tissues and to exert a broad spectrum of pharmacological effects. Despite this attention, however, relatively little is known about the actual physiologic significance of this substance. One area in which the biologic importance of VIP is beginning to be defined is reproduction. Considerable evidence suggests that VIP may play a role in both central and peripheral reproductive processes. A number of investigators have concluded, for example, that VIP is a probable PRLreleasing factor in rats, monkeys, and man [2, 6, 14, 19, 22, 30-32, 34]. Others have suggested that VIP may function as a neurotransmitter in the female genital tract itself, and serve to influence the contractility of the muscular elements of the vasculature, as well as the ovarian follicle, the oviduct and the uterus [4, 7, 9, 10, 21, 23, 25, 33]. The observation reported by this laboratory that VIP infused into rabbits markedly elevated plasma levels of P, indicated that VIP might also play a role in the regulation of ovarian steroid secretion. The data summarized in this report, as well as that reported previously by Fredericks et al. [8], suggest, however, that this effect upon P is not related to reproductive processes per se. Any direct involvement of VIP in the central regulation of P secretion, particularly as a component of reproductive function, would most likely involve VIP effects upon the anterior pituitary hormones, LH, F S H , and PRL. There is little evidence that this is occurring in the rabbit.
TABLE 1 PLASMA CORTISOL (p,g/dl)*
Time (hr)
75 pmol VIP
Vehicle
Intact 0
10.5 ± 1.3 (8)
1
19.2 ± 1.5 (8)**
12.0 ± 0.6 (6) 10.4 -+ 1.2 (6)
Ovariectomized (OVX) 0 1
13.0 ± 0.4 (9) 21.8 ± 2.1 (9)**
Not Done
Ovariectomized/Adrenalectomized (OVX/ADX) 4.3 ± 0.5 (9) 6.6 ± 1.0 (9)
Not Done
*Values are mean - S.E.M.; number of animals in parentheses. VIP (75 pmoi/kg per min) or vehicle (lactated ringers) was infused intravenously for 1 hr (T+0 to T+ 1). **Indicates values significantly different (p<0.01) from corresponding preinfusion values. OVX/ADX pre- and post-infusion values are significantly different (p<0.001) from corresponding values of both intact and OVX.
Progesterone-stimulating infusions of VIP are not associated with increased secretion of F S H or LH, in fact a slight inhibition of F S H was noted. Also, if the P increases in the rabbit were the result of a significant increase in LH, ovulation and follicular luteinization would have been expected. This did not occur. Other investigators have shown similarly that infused VIP does not increase LH or F S H in rats or humans [22,34]. By the same token, Fredericks et al. [8] have previously shown that the effect of VIP upon P in the rabbit is not associated with any significant change in plasma PRL levels, nor is it affected by a PRL-lowering treatment with bromocriptine. Not only are the gonadotropins apparently not involved, but the ovaries themselves do not appear to be the source of the VIP-stimulated P. The observation that OVX has little effect upon the P response, while it is abolished by ADX, suggests that the source of the P is, in fact, the adrenal gland. Although substantial amounts of P are synthesized by both the adrenals and ovaries in the rabbit [27], VIP appears to stimulate primarily the adrenal component. The effect of VIP upon adrenal secretion is also indicated by the observation that P only increased when cortisol significantly increased, such as in the intact and OVX animals. In the OVX/ADX animals, in which no significant increase in cortisol accompanied the VIP infusion, P did not increase. The mechanism of this VIP effect, however, remains unknown. Any central effect upon the adrenal cortex would be mediated by increased pituitary ACTH release. To date, however, it is not at all clear whether VIP has any significant effects upon ACTH release, either in vivo or in vitro. Experiments conducted in intact animals have been contradictory. Bataille et al. [1] infused VIP into normal humans and observed a significant increase in serum ACTH. Ottesen et al. [22], on the other hand, in similar experiments found no increase in plasma cortisol. When infused into normal rats intracisternally, V1P increased ACTH release, but not when
VIP S T I M U L A T I O N O F A D R E N A L P R O G E S T E R O N E infused intravenously [13]. VIP has been shown to produce a dose-dependent stimulation of A C T H release from a line of human pituitary tumor cells [20], but no significant change in the A C T H released from purified rat pituitary cells [28] or incubated rat pituitary tissue [13]. VIP produces a stimulation of steroidogenesis in cultured mouse adrenal cortical tumor cells as great as that produced by A C T H [15]. These effects, however, require high VIP doses and appear to be exerted through receptors distinct from those of A C T H [3]. VIPergic nerve fibers are present in the adrenal medulla and cortex of the rat [12] and could conceivably exert a local effect upon P secretion. It remains to be determined whether the VIP effects upon P secretion reflect a direct effect upon the pituitary-adrenal axis, and hence may suggest some physiological role for VIP in the regulation of adrenal secretion, or whether this increase in P merely accompanies a generalized activation of adrenal mechanisms resulting from VIP effects upon other biologic processes. Interestingly, a significant amount o f P remained in the plasma of OVX/ADX animals. The source of this P, however, has yet to be determined. It is unlikely that it is se-
209
creted by the adrenal tissue remaining postoperatively in some animals, since the amount of this tissue was small (calculated to be less than 2-3% of an intact gland) and was shown to be unable to support the VIP effect upon either P or cortisol. In addition, no positive correlation could be established between the amount of adrenal tissue noted at autopsy and the plasma levels of P. It is more likely that the P is coming from some other tissue, perhaps from the uterus. Overstrom and Black (1980; personal communication, 1984) have shown that plasma P remains after OVX/ADX in rabbits and that rabbit endometrial cells can synthesize P in culture. This suggests that in vivo the rabbit uterus may contribute to overall levels of circulating P.
ACKNOWLEDGEMENTS The authors would like to thank Dr. Thomas Mills, Medical College of Georgia, for his generous support of this project in contributing the LH and FSH assays. We also thank Judy Kameoka for her diligent technical assistance. This work was supported in part by the South Carolina State Appropriation for Biomedical Research.
REFERENCES I. Bataille, D., J. N. Talbot, G. Miihaud, V. Mutt and G. Rosselin. Effet du peptide intestinal vasoactif (VIP) sur la secretion de prolactine chez l'homme. CR Acad Sci (Paris) 292: 311-313, 1981. 2. Besson, J., W. H. Rotsztein and D. Bataille. Involvement of VIP in neuroendocrine functions. In: Vasoactive Intestinal Polypeptide, edited by Sami I. Said. New York: Raven Press, 1982, pp. 253-262. 3. Birnbaum, R. S., M. Alfonzo and J. Kowal. Vasoactive intestinal peptide- and adrenocorticotropin-stimulated adenylcyclase in cultured adrenal tumor cells: Evidence for a specific vasoactive intestinal polypeptide receptor. Endocrinology 1116: 12701275, 1980. 4. Clark, K. E., E. G. Mills, S. J. Stys and A. E. Seeds. Effects of vasoactive polypeptides on the uterine vasculature. Am J Obstet Gynecol 139: 182-188, 1981. 5, Fahrenkrug, J. VIP as a neurotransmitter in the peripheral nervous system. In: Vasoactive Intestinal Polypeptide, edited by Sami I. Said. New York: Raven Press, 1982, pp. 361-372. 6. Frawley, L. S. and J. D. Neill. Stimulation of prolactin secretion in Rhesus monkeys by vasoactive intestinal polypeptide. Neuroendocrinology 33: 7%83, 1981. 7. Fredericks, C. M. and S. H. Ashton. Effect of vasoactive intestinal polypeptide (VIP) on the in vitro and in vivo motility of the rabbit reproductive tract. Fertil Steril 37: 845-850, 1982. 8. Fredericks, C. M., L. E. Lundquist, R. S. Mathur, S. H. Ashton and S. C. Landgrebe. Effects of vasoactive intestinal polypeptide upon ovarian steroids, ovum transport, and fertility in the rabbit. Biol Reprod 28: 1052-1060, 1983. 9. Helm, G., B. Ottesen, J. Fahrenkrug, J. J. Larsen, C. Owman, N. O. Sjoberg, B. Stolberg, F. Sundler and B. Walles. Vasoactive intestinal polypeptide (VIP) in the human reproductive tract: Distribution and motor effects. Biol Reprod 25: 227-234, 1981. 10. Helm, G., S. Hakanson, S. Leander, C. Owman, N. O. Sjoberg and B. Sporrong. Neurogenic relaxation mediated by vasoactive intestinal polypeptide (VIP) in the isthmus of the human fallopian tube. Regal Pept 3: 145-153, 1982. II. Hokanson, R., F. Sundler and R. Uddman. Distribution and topography of peripheral VIP nerve fibers: Functional implications. In: Vasoactive Intestinal Polypeptide, edited by Sami I. Said. New York: Raven Press, 1982, pp. 121-144.
12. Hokfelt, T., M. Schultzberg, J. M. LundbeI'g, K. Fuxe, V. Mutt, J. Fahrenkrug and S. I. Said. Distribution of vasoactive intestinal polypeptide in the central and peripheral nervous systems as revealed by immunocytochemistry. In: Vasoactive Intestinal Polypeptide, edited by Sami I. Said. New York: Raven Press, 1982, pp. 65-90. 13. Itoh, S., R. Hirota and G. Katsuura. Effect of cholecystokinin octapeptide and vasoactive intestinal polypeptide on adrenocortical secretion in the rat. Jpn J Physiol 32: 553-560, 1982. 14. Kato, Y., Y. Iwasaki, J. Iwasaki, H. Abe, N. Yanaihara and H. Imura. Prolactin release by vasoactive intestinal polypeptide in rats. Endocrinology 103: 554-558, 1978. 15. Kowal, J. VIP effects on adrenocortical cell functions. In: Vasoactive Intestinal Polypeptide, edited by Sami I. Said. New York: Raven Press, 1982, pp. 277-284. 16. Marley, P. and P. Emson. VIP as a neurotransmitter in the central nervous system. In: Vasoactive Intestinal Polypeptide, edited by Sami I. Said. New York: Raven Press, 1982, pp. 341360. 17. Mathur, R. S., S. Landgrebe and H. O. Williamson. Progesterone, 17-hydroxyprogesterone, estradiol, and estriol in late pregnancy and labor. Am J Obstet Gynecol 136: 25-30, 1980. 18. Mills, T. M. and R. J. Gerardot. Dissociation of copulation from ovulation in pregnant rabbits. Biol Reprod 30: 1243-1252, 1984. 19. Nicosia, S., A. Spada and G. Giannattasio. Effects of vasoactive intestinal polypeptide on the pituitary gland. In: Vasoactive Intestinal Polypeptide, edited by Sami I. Said. New York: Raven Press, 1982, pp. 263-275. 20. Oliva, D., S. Nicosia, A. Spada and G. Giannattasio. VIP stimulates ACTH release and adenylate cyclase in human ACTHsecreting pituitary adenomas. Eur J Pharmacol 83: 101-105, 1982. 21. Ottesen, B. and J. Fahrenkrug. Effect of vasoactive intestinal polypeptide (VIP) upon myometrial blood flow in non-pregnant rabbit. Acta Physiol Scand 112: 195-201, 1981. 22. Ottesen, B., A. N. Anderson, T. Gerstenberg, H. Ulrichsen, T. Manthorpe and J. Fahrenkrug. VIP stimulates prolactin release in women. Lancet 2: 696, 1981.
210 23. Ottesen, B., J. J. Larsen, J. Fahrenkrug, M. Stjernquist and F. Sundler. Distribution and motor effects of VIP in female genital tract, Am J Physiol 240: E32-36, 1981. 24. Ottesen, B., H. Ulrichsen, J. Fahrenkrug, J. J. Larsen, G. Wagner, L. Schierup and F. Sondergaard. Vasoactive intestinal polypeptide and the female genital tract: Relationship to reproductive phase and delivery. Am J Obstet Gynecol 143: 414-420, 1982. 25. Ottesen, B., F. Sondergaard and J. Fahrenkrug. Neuropeptides in the regulation of female genital smooth muscle contractility. Acta Obstet Gynecol Scand 67: 591-592, 1983. 26. Ottesen, B., T. Gerstenberg, H. Ulrichsen, T. Manthorpe, J. Fahrenkrug and G. Wagner. Vasoactive intestinal polypeptide (VIP) increases vaginal blood flow and inhibits uterine smooth muscle activity in women. Eur J Clin Invest 13: 321-324, 1983. 27. Overstrom, E. W. and D. L. Black. Effect of ovariectomy and adrenalectomy on peripheral plasma concentrations of estradiol and progesterone in the rabbit. Biol Reprod 22: Suppl, 134A (Abstract), 1980. 28. Rotsztejn, W. H., L. Benoist, J. Besson, G. Beraud, M. T. Bluet-Pajot, C. Kordon, G. Rosselin and J. Duval. Effect of vasoactive intestinal polypeptide (VIP) on the release of adenohypophyseal hormones from purified cells obtained by unit gravity sedimentation: Inhibition by dexamethasone of VIP-induced prolactin release. Neuroendocrinology 31: 282286, 1980.
FREDERICKS ET AL. 29. Said, S. I. and V. Mutt. Polypeptide with broad biological activity: Isolation from small intestine. Science 160: 1217-1218, 1970. 30. Said, S. I. and J. C. Porter. Vasoactive intestinal polypeptide release into hypophyseal portal blood. Life Sci 24: 227-230, 1979. 31. Shumatsu, A., Y. Kato, N. Matsushita, H. Katakami, B. Yanalhara and H. Imura. Immunoreactive vasoactive intestinal polypeptide in rat hypophyseal portal blood. Endocrinology 108: 395-398, 1981. 32. Shumatsu, A., Y. Kato, H. Ohta, K. Tojo, Y. Kabayama, T. Inoue, N. Yanaihara and H. Imura. Involvement of hypothalamic vasoactive intestinal polypeptide (VIP) in prolactin secretion induced by serotonin in rats. Proc Exp Biol M5: 414-416, 1984. 33. Strom, C., J. M. Lundberg, H. Ahlaman, A. Dahlstrom, J. Fahrenkrug and T. Hokfelt. On the VIPergic innervation of the uterotubal junction. Acta Physiol Scand I l l : 213-215, 1981. 34. Vijayan, E., W. K. Samson, S. I. Said and S. M. McCann. Vasoactive intestinal polypeptide: Evidence for a hypothalamic site of action to release growth hormone, luteinizing hormone, and prolactin in conscious ovariectomized rats. Endocrinology 104: 53-57, 1979.