Normal Female Rats Research Articles

Purification of functional lactotrophs and somatotrophs from female rats using fluorescence-activated cell sorting

As the secretory granules of anterior pituitary cells fuse with the cell surface, there would appear to be sufficient hormone present on the cell surface to be labelled by polyclonal hormone antibodies and thus analysed by flow cytometry. We have therefore applied fluorescence-activated cell sorting to these labelled pituitary cells. Percentage purity and depletion of other cell types was assessed by immunocytochemistry and the reverse haemolytic plaque assay (RHPA). Results demonstrate that fluorescence-activated cell sorting allows almost complete purification of functional lactotrophs and somatotrophs to 96.7 +/- 1.7 (S.E.M.)% and 98 +/- 1.0% respectively by immunocytochemistry, and to 95.8 +/- 1.1% and 97 +/- 0.8% respectively by RHPA. Depletion of other anterior pituitary cell types to less than 2% was demonstrated by both immunocytochemistry and RHPA. Fluorescence-activated cell sorting to this degree of purity was routinely possible with cell yields of 91 +/- 3.4%. To obtain such purity/depletion, it was necessary to use specific antisera of high titre, at concentrations which ensured maximal cell-surface labelling associated with maximal stimulation of hormonal secretion by the appropriate hypothalamic stimulatory factor. Separating cells on the basis of the intensity of prolactin cell-surface labelling demonstrated a low level of binding of the prolactin antibody to gonadotrophs (but not of sufficient fluorescence intensity to be sorted into the prolactin enriched population), raising the possibility of prolactin receptors on gonadotrophs. We were unable to demonstrate the presence of mammosomatotrophs in the normal female rat, since purified lactotrophs did not contain or secrete GH nor did purified somatotrophs contain or secrete prolactin.

Assessment of urinary rat β 2-microglobulin by enzyme immunoassay

An enzyme immunoassay (EIA) was developed using unlabelled and peroxidase-labelled rabbit antibodies to assess urinary rat β 2-microglobulin (β 2-m) excretion. It consisted in the adsorption of rabbit anti-rat β 2-m immunoglobulin to a polystyrene surface, the addition of β 2-m samples or standard and the addition of peroxidase-labelled rabbit anti-rat β 2-m immunoglobulin. After addition of hydrogen peroxide and o-phenylenediamine, the enzyme activity of the solid phase was measured photometrically at 490 nm. Analytical recovery of pure β 2-m in urine was 1029%. From determinations made on normal female and male rats, the ranges of 24-h urine β 2-m individually excreted were found to be 0.84–4.77 and 3.7–17.3 μg, respectively. The means ± SEM were 2.49 ± 0.17 and 10.2 ± 0.55 μg. EIA was of value in evidencing the tubule-damaging properties of sodium chromate and hexachloro-1,3-butadiene in rats.

Role of postnatal androgens in sexual differentiation of the lordosis‐inhibiting effect of central injections of cholecystokinin

The neuropeptide cholecystokinin (CCK) inhibits lordosis behavior when infused into the ventromedial nucleus of the hypothalamus (VMN) of female rats and has no effect when infused into the VMN of male rats. To test whether this sex difference develops under the control of perinatal steroids, male rats were castrated or given sham surgeries within 3 h of birth and female rats were injected with either 0 or 100 micrograms testosterone propionate on postnatal day 5. As adults, these rats were castrated as necessary, implanted with unilateral cannulae directed at the VMN, and tested for their ability to display female sexual behavior and to respond to CCK. Neonatal castration of males prevented defeminization of this response. When treated with 5 micrograms estradiol benzoate (EB), neonatally castrated males showed both lordosis behavior and a profound inhibition of that behavior after infusions of CCK. Neonatally castrated males did not display lordosis behavior when treated with 2 micrograms EB. Control males showed no lordosis behavior and, therefore, no response to CCK. Both doses of EB induced lordosis behavior in neonatally androgenized females. Significantly, these neonatally androgenized females were less responsive to CCK's inhibition of lordosis and were also anovulatory. These results imply that androgens alter the development of CCK responsive circuits as well as defeminize cyclic gonadotropin release. Levels of 125I-sCCK-8 binding in the VMN were correlated closely with an individual's ability to respond to sCCK-8. In summary, the inhibition of female sexual behavior caused by exogenously administered CCK in normal adult female rats appears to be controlled at least partially by levels of CCK receptors in the VMN and to differentiate under the control of perinatally present testosterone.

The capacity to develop maternal behavior is enhanced during aging in rats

The induction of maternal behavior (MB) in response to stimulation by pups was studied in aged rats (19–20 months old). We used virgin female rats, neon androgenized female rats and male rats. Both groups of female rats showed a constant estrous vaginal smear. Maternal responsiveness was compared with that of young rats (3–4 months old). Normal and androgenized female aged rats showed a very high percentage of immediate maternal responsiveness and 100% of the rats were fully maternal within 24 hr of testing. The percentage of cyclic and androgenized young rats showing MB were significantly lower. Chronic ovariectomy performed 17 months before testing but not acute ovariectomy abolished MB. Estrogen treatment (5 μg 15 hours before pup presentation) to chronically ovariectomized aged rats was not sufficient to reestablish significantly t capacity of the normal female aged rats to become short-latency maternal. Young and aged male rats showed no difference in maternal responsiveness to the presence of foster pups. The percentage of maternal aged male rats was significantly lower than that of the normal and androgenized aged female rats, whereas young male and female rats showed a similar level of MB, indicating a sex difference in the development of MB with age. In conclusion the high percentage of rats becoming maternal and the short-latency maternal responsiveness in aged female rats appears to be the result of a prolonged estrogen and/or prolactin stimulation.

Cyclooxygenase-dependent mediators of renal hemodynamic function in female rats

Previous studies have revealed a sex-dependent difference in response to cyclooxygenase inhibition in anesthetized male and female rats. Female rats have shown an unexpected vasodilation in response to prostaglandin (PG) inhibition. The present studies were designed to further investigate the sex-dependent role of the PG system in the control of normal renal hemodynamics in female Munich-Wistar rats. Renal hemodynamic studies were performed on anesthetized female rats before and during acute cyclooxygenase inhibition using a variety of protocols. One group underwent subacute unilateral renal denervation. A separate group was chronically catheterized and plasma catecholamines were measured during the awake state and then after anesthesia under the euvolemic protocol. Another group was administered the angiotensin II blocker, saralasin, before and during cyclooxygenase inhibition. In a final group, flow to the distal nephron was interrupted via placement of a wax block into the late proximal tubule to determine the role of distal nephron flow and tubuloglomerular feedback in the glomerular response to cyclooxygenase inhibition. It was determined that neither the wax block nor saralasin administration attenuated the vasodilatory response observed in normal female rats due to cyclooxygenase inhibition; however, subacute unilateral renal denervation completely blocked the vasodilatory response to PG inhibition in these female Munich-Wistar rats. Plasma catecholamines were found to be similar whether awake or under anesthesia. These studies indicate the importance of the adrenergic system in modulating PG production in the acutely anesthetized intact female rat.

Somatostatin and its Physiological Significance in Regulating the Episodic Secretion of Growth Hormone in the Rat

Somatostatin (SS) is a powerful inhibitor of growth hormone (GH) secretion both in vitro and in vivo, and is generally regarded as having a negative influence on GH secretion and growth. In the conscious rat, the GH secretory pattern, the response to repeated injections of GH-releasing hormone (GHRH), and the feedback mechanism by which GH regulates its own release are all sexually dimorphic and probably reflect a sexually dimorphic pattern of SS release. Prolonged infusions of SS initially block GHRH-induced GH release but responses begin to break through with time. Withdrawal of SS induces a rebound release of GH in vivo, largely dependent on the release of endogenous GHRH. Prolonged exposure to intermittent infusions of SS in normal female rats produces a paradoxical growth response by inducing regular peaks of GH secretion whereas continuous infusions of SS do not. This response is GH-dependent, and is not due to other effects of SS since intermittent infusions of SS do not increase growth in dwarf rats which are deficient in pituitary GH. The sex differences in GH secretion are also reflected in the expression of several GH-dependent liver enzymes, one of which, carbonic anhydrase III, responds to manipulations of the endogenous secretory pattern by SS in both normal and dwarf rats, and appears to be sensitive to differences in basal, rather than peak, GH levels.

Presence of Immunoreactive Vasoactive Intestinal Polypeptide in Anterior Pituitary of Normal Male and Long Term Estrogen-Treated Female Rats: A Light Microscopic Immunohistochemical Study*

The presence and synthesis of vasoactive intestinal polypeptide (VIP) has been demonstrated in the rat anterior pituitary. It was recently confirmed that immunoreactive VIP is present in the anterior pituitary, and in a thyroid-deficient state, VIP could be detected by light microscopic immunohistochemistry. It has also been suggested that VIP plays a stimulatory role in PRL secretion. To gain more detailed information on the localization of VIP and the conditions that alter the synthesis of VIP, we examined VIP immunoreactivity using immunohistochemistry in pituitaries of normal male and cycling female rats and in those states in which PRL secretion was enhanced (pregnancy, lactation, long term estrogen treatment, and pituitary implanted under kidney capsule of normal or estrogen-treated female rats). In situ, implanted and cultured pituitary cells were stained for VIP immunoreactivity using the peroxidase-antiperoxidase method. VIP immunoreactivity was observed in about 45% of the male rats, in all estrogen-treated female rats, in the implanted pituitaries under the kidney capsule (three of five from estrogen-treated and one of five from intact females, respectively), and in the pituitary cell cultures derived from estrogen-treated rats. Using a double labeling procedure we have also observed PRL immunoreactivity in a small population of the VIP-positive cells. These results suggest a positive regulatory role of estrogen in expression of the VIP gene. The physiological and pathophysiological significance of VIP in PRL secretion, however, remains to be clarified.

X‐chromosome monosomy in the myelin‐deficient rat mutant

We have identified three examples of female Wistar rats exhibiting the tremor and seizures characteristic of the X-linked myelin deficiency (md) mutation, which is ordinarily seen only in males. Cytogenetic study of two of these animals has shown them to have 41 chromosomes instead of the normal 42. The missing chromosome was identified as an X chromosome by G-banding analysis. These animals thus have an XO genotype comparable to that in Turner's syndrome. Anatomically, one of the animals, which was studied in detail, showed no abnormality of the uterus, and the ovaries, although somewhat smaller than normal, were histologically indistinguishable from those in a normal female rat. No evidence of endocardial fibroelastosis was detected, nor was there any anomaly of the aorta. The myelin deficiency in the central nervous system was comparable to that in hemizygous mutant male rats. XO monosomy in the Wistar rat thus has little effect on phenotype and is more comparable to that in mice than to Turner's syndrome in man. The myelin-deficient rat is useful for studies of X-chromosome monosomy since XO females can readily be identified by the neurological syndrome characteristic of the md mutation.

The episodic secretory pattern of growth hormone regulates liver carbonic anhydrase III. Studies in normal and mutant growth-hormone-deficient dwarf rats

Carbonic anhydrase III (CAIII) occurs in male rat liver at concentrations twenty times those in the female, and is sensitive to the pattern of growth hormone (GH) release. Males release GH episodically and have high concentrations of CAIII; females produce GH in a more continuous fashion and have lower CAIII levels. In normal female rats, the endogenous GH secretory pattern was masculinized, either by regular injections of GH-releasing factor (GRF) or by intermittent infusions of somatostatin (90 min on/90 min off). Both treatments induced regular GH pulses and stimulated growth, but only intermittent somatostatin infusions raised CAIII levels (controls, 1.5 +/- 0.5; somatostatin-treated, 9.0 +/- 2.9 micrograms/mg; means +/- S.D.). GRF pulses (4 micrograms every 4 h) did not however raise CAIII levels (controls 1.8 +/- 0.5; GRF-treated 1.4 +/- 0.4 micrograms/mg). Surprisingly, hepatic CAIII is also sexually dimorphic (males, 18.8 +/- 3; females, 2.22 +/- 0.4 micrograms/mg) in a GH-deficient dwarf rat strain which has low plasma GH levels without 3-hourly GH peaks. Intermittent somatostatin infusions in female dwarf rats partially masculinized hepatic CAIII, an effect reduced by co-infusion with GRF. This CAIII response was not secondary to growth induction, since neither somatostatin nor GRF stimulated growth in dwarf rats, and pulses of exogenous GH stimulated growth in female dwarfs without masculinizing CAIII levels. Furthermore, continuous GH infusion in male dwarf rats partially feminized hepatic CAIII levels (to 9.1 +/- 2.4 micrograms/mg), whereas infusions of insulin-like growth factor-1, which induced the same body weight gain, did not affect hepatic CAIII (20.8 +/- 6 micrograms/mg). These results show that hepatic CAIII expression is highly sensitive to the endogenous GH secretory pattern, independent of growth. They also implicate the low basal GH levels between pulses, rather than the peak GH levels, as the primary determinant of the sexually dimorphic hepatic CAIII expression in the rat.

Fetal cortical transplants reduce motor deficits resulting from neonatal damage to the rat's frontal cortex

Motor deficits in the execution of grasping movements of the right forelimb were compared in normal female Wistar rats, in animals which sustained a neonatal lesion of the left frontal cortex and in animals which received immediately after the lesion a transplant obtained from the frontal cortex of E16 embryos. Behavioral testing was carried out from postnatal day 48 (D48) to D108. The animals were placed at the center of a circular wire grid which was turned upside down so that they hung by their 4 paws at a distance of 40 cm above the floor. The precision of grasping movements of the right limb and the number of falls were recorded during a 1 min session of active moving across the grid. The lesioned subjects were most impaired on both motor indices whereas the grafted animals although performing slightly poorer than the controls were, however, less impaired than the lesioned animals. Fetal cortical transplants, therefore, seem to promote functional recovery from neonatal cortical damage.

Radioiodinated ligands for the estrogen receptor: Tissue distribution of 17α-[ 125I]iodovinylestradiol derivatives in normal and tumor-bearing adult female rats

A series of three 125I-labeled 17α-iodovinylestradiol derivatives previously demonstrated to have a selectivity for estrogen receptor tissues in immature female rats were selected for further evaluation in adult female rats. In normal adult female rats, the iodovinyl analogs of moxestrol (IVβME 2) and moxestrol-3-O methyl ether (IVβME 2-3-OMe) demonstrated high uterine uptake (0.296–0.437 and 0.135–0.199%ID-kg/g) and selectivity (10–18:1) over the 6 h time period. Subsequent evaluation in two tumor models indicated that [ 125I]VβME 2 also possessed the highest tumor uptake and selectivity in the adult female rats bearing the estrogen responsive 9,10-dimethyl-1,2-benzanthracene(DMBA)-induced mammary tumors and that this was receptor mediated. An estrogen independent tumor, the transplanted Walker 256 mammary adenocarcinoma, showed no selectivity of radioligand uptake compared with nontarget tissues. The results suggest the applicability of this agent to the in vivo detection and characterization of estrogen responsive tumors in man.

Determination of regional blood flow in abdominal organs and other structures in normal female rats and in rats with TAA-induced chronic liver injury using 99mTc labelled HSA-microsphere technique

A method for simultaneous determination of cardiac output and regional blood flow distribution in a chronically instrumented, unrestrained rat preparation for different experimental conditions (i.e. conscious, free movement; general anesthesia) and in an experimental chronic liver injury model is described. The use of a modified radioactive microsphere reference sample method using 99mTc labelled HSA-microspheres provides valid measurements of cardiac output [255 +/- 21.6 ml/(min.kg b.wt.)] and of the determined blood flow rates of abdominal organs (with separate determination of arterial and portal-venous hepatic blood flow rates; the latter by means of arterial blood flow measurement of the gastrointestinal tract and the spleen), the myocard, the adrenals, the kidneys, and various brain regions. Furthermore, it is demonstrated that this measuring approach with chronical preparation can also advantageously be used in pharmacological or pathogenetical studies, especially because of the simple measuring equipment and the comparatively low costs that offer a broader application.

Gonadal Steroid Regulation of Substance P (SP) and SP-Encoding Messenger Ribonucleic Acids in the Rat Anterior Pituitary and Hypothalamus*

Substance P-like immunoreactivity (SP-LI) is present in the rat anterior pituitary (AP) and in hypothalamic neurons that may be involved in the control of AP secretion and/or reproductive function. The presence of multiple SP-encoding mRNAs and tachykinin peptides and their regulation by steroid hormones were examined in APs and hypothalami from normal, gonadectomized, and steroid-treated male and female rats. SP-encoding mRNAs were identified by nuclease protection assays of RNA, and tachykinin peptides were identified by combined HPLC-RIA of tissue extracts, beta- and gamma-preprotachykinin (PPT) mRNAs and SP, neurokinin A, and neuropeptide gamma peptides were identified in the AP. The alpha-, beta-, and gamma-PPT mRNAs and SP, neurokinin A, neuropeptide gamma, neuropeptide K, and neurokinin B peptides were present in hypothalamic tissue. Previous studies have established that in the AP, SP is differentially regulated by gonadal steroids; estrogen decreases and androgen increases AP SP. Steroid effects were further analyzed in experiments using RIAs to measure SP levels in the AP and median eminence (ME) of steroid- and oil-treated gonadectomized rats. To assess whether steroids alter steady state PPT mRNA levels and presumably SP synthesis in these tissues, potential effects on AP and hypothalamic SP-encoding mRNAs were determined. Ovariectomized rats treated for 10 days with estradiol benzoate showed a 50% decrease in AP SP and a 90% decrease in AP beta- and gamma-PPT mRNAs compared to ovariectomized oil-treated controls. Estradiol benzoate replacement had no effect on SP levels in the isolated ME, but did cause a 50% increase in alpha-, beta-, and gamma PPT mRNAs in the hypothalamus. Although there was no significant effect of testosterone propionate on AP SP levels in castrated males, 10 days of testosterone propionate replacement did cause a significant increase in beta- and gamma PPT mRNAs in the AP. No androgen effects were seen on either ME SP or hypothalamic SP-encoding mRNAs. These data demonstrate that estrogen up-regulates SP-encoding mRNAs in the hypothalamus, whereas it down-regulates SP-encoding mRNAs in the pituitary. These results implicate SP and other tachykinins derived from the SP gene as steroid-regulated modulators of AP secretion and possibly reproductive function.

Modeling cortical cataractogenesis: IX. Activity of vitamin E and esters in preventing cataracts and gamma-crystallin leakage from lenses in diabetic rats.

Normal and streptozotocin diabetic female Wistar rats were given vitamin E in the diet as the tocopherol, acetate, or succinate form (2,850 IU/kg food). At the end of 6 weeks, the rats were examined for weight gain or loss, general body condition, and cataracts. At sacrifice, blood was collected for measurement of serum glucose, and gamma-crystallin levels were measured in aqueous and vitreous humors using a radioimmunoassay. One lens was homogenized in 8 M guanidinium chloride for ATP analysis. In normal rats, gamma-crystallin was detected in both aqueous and vitreous humors, with the higher concentration in the vitreous humor. Diabetes caused a sixfold increase in gamma-crystallin in both the aqueous and vitreous humors. Diabetes also led to a significant worsening in general body condition, loss of body weight, formation of cataracts, and decrease in lens ATP levels. Addition of vitamin E and vitamin E succinate, but not vitamin E acetate, to the diet resulted in reduction of gamma-crystallin leakage into the vitreous humors and an increase in body weight. There was no improvement noted for the lens ATP levels, the general body condition, or visual cataract score. Neither streptozotocin-induced diabetes nor vitamin E in the diet appeared to affect the weight of the lenses.

Differential secretory rhythm of growth hormone controls the number of hepatic epidermal growth factor receptors in the rat.

The effect of human GH (hGH) on hepatic epidermal growth factor (EGF) receptors in the rat was investigated. Continuous administration of hGH through an osmotic minipump, mimicking the female pattern of GH secretion, to normal male rats reduced the binding of 125I-labelled EGF to hepatic membranes to the normal female levels. The same treatment of hGH applied to hypophysectomized males had no apparent effect on EGF binding. Intermittent s.c. administration of hGH twice a day (every 12 h), mimicking the male pattern of GH secretion, to hypophysectomized male and/or normal female rats, caused a significant increase in EGF binding to the levels of normal male rats. Scatchard analysis of the binding data clearly showed that the change in EGF binding was due to a change in the number of EGF receptors. The results on the affinity labelling and phosphorylation of EGF receptors were in good agreement with those showing differences in the number of EGF receptors among the experimental groups. These results indicate that the number of hepatic EGF receptors in the rat is regulated by the differential secretory rhythm of pituitary GH between the sexes.

Interaction of Human Growth Hormone and Gonadotrophins on the Function of Rat Ovaries

Hypophysectomized and normal female rats were used for studying the interaction of human growth hormone and gonadotrophins in stimulating the rat ovary. Groups of rats were injected with increasing doses of gonadotrophins (Pergonal or Metrodin) in addition to human growth hormone (Norditropin) or placebo. In the test for follicle stimulating hormone activity the weight of the ovaries was recorded. In the hypophysectomized rats Norditropin and Pergonal or Metrodin had a synergistic stimulating effect on the ovary, resulting in a greater number and a larger follicle size. Norditropin had a slight stimulatory effect of its own. In the ovarian ascorbic acid depletion test for luteinising hormone activity Norditropin had a significant effect of its own. In combination with Pergonal an additive effect was seen. The effect of Norditropin was observed in hypophysectomized rats only.

Galactose as a regulatory factor of its own metabolism by rat liver

As part of a series of studies to assess the regulation of hepatic galactose-metabolizing enzymes, galactokinase, galactose-1-phosphate uridyltransferase, and UDPgalactose-4-epimerase, the effect of feeding a high galactose-containing diet to normal adult and pregnant female rats was examined. Sixteen days of galactose exposure of adult virgin females produced a different response in the specific activity of each of the enzymes, that of galactokinase being lower, transferase higher, and epimerase transiently elevated. Galactose feeding increased the specific activity of transferase in pregnant rat liver above the elevated level that already exists in the pregnant state but failed to influence the enzyme of the developing fetal liver. Galactose added to liver homogenates did not activate transferase. The increased activity in liver of adult, fed animals was not associated with a change in isoenzyme patterns examined by isoelectric gel electrophoresis but was characterized on kinetic analysis by an increase in Vmax for UDPglucose. The changes in enzyme specific activity in liver of animals fed galactose appear to have physiologic significance because hepatocytes isolated from galactose exposed livers take up more galactose and convert more to glucose and lactate than cells from control animals.

Androgen regulation of rat renal angiotensinogen messenger RNA expression.

Renal angiotensinogen (ang-n) mRNA concentration in the male WKY rat increases significantly during puberty. Furthermore, renal angiotensinogen mRNA level in the adult female WKY rat is considerably lower than in the male. The present study investigates the role of androgen in differential renal ang-n mRNA expression. Northern and slot blot analyses with alpha-32P labeled ang-n cDNA (pRang 3) demonstrated that castration lowered ang-n mRNA levels in the male kidney by greater than or equal to 60% compared with control, suggesting that androgen may be involved with renal ang-n gene regulation. Moreover, male WKY rats castrated as weanlings and normal adult female WKY rats each implanted with testosterone displayed significant (P less than 0.05) increases in renal ang-n mRNA levels. Our observations, taken together with previous reports that androgen influences proximal tubule morphology and the tubular expression of transport proteins (e.g., Na+/H+ antiporter), may have important physiological implications for understanding the relationship between androgen and angiotensin in the regulation of tubular function.

Estrogen stimulates gonadotropin-releasing hormone release from rat hypothalamus independently through catecholamine and histamine in vitro

Estradiol is known to stimulate gonadotropin-releasing hormone release from the rat medio-basal hypothalamus. Studies were made in an in vitro perifusion system on whether catecholamine and/or histamine was involved in estradiol-induced GnRH release. Normal cycling female rats were decapitated in diestrus II and their medio-basal hypothalami were combined and, perifused with Earl's balanced salt solution containing 0.01% bovine serum albumin bubbled with 95% O2 and 5% CO2. The levels of norepinephrine, dopamine, and histamine and of GnRH in the effluent were measured by HPLC and radioimmunoassay, respectively. Administration of 10(-6) mol/l estradiol resulted in releases of norepinephrine, dopamine, histamine and GnRH at levels of 98, 70, 91 and 288%, respectively, of initial values. Administration of 10(-6) mol/l norepinephrine or dopamine resulted in no increase in histamine release, and administration of 10(-6) mol/l histamine did not increase release of norepinephrine or dopamine. These data suggest that estradiol stimulates the releases of GnRH, catecholamine and histamine from the rat medio-basal hypothalamus, and that it increases GnRH release independently through catecholamine and histamine. As we found previously that norepinephrine or histamine stimulates GnRH release from the medio-basal hypothalamus, we conclude that estradiol stimulates releases of norepinephrine and histamine, resulting in GnRH release from the medio-basal hypothalamus.

Characterization of 17-β-Estradiol-dependent and -independent Somatostatin Receptor Subtypes in Rat Anterior Pituitary

Abstract Previous studies from this laboratory showed that treatment with 17-beta-estradiol (E2) caused an acquisition of inhibitory effect of somatostatin (SRIF) on prolactin release with an increased number of SRIF-binding sites in the rat anterior pituitary. The aim of this study was to characterize the E2-dependent SRIF receptor in comparison with the E2-independent one, which was expressed in ovariectomized rats. The following observations were obtained: 1) both of the E2-dependent and E2-independent SRIF receptors, measured with 125I-Tyr11-SRIF as a radiolabeled ligand, were enriched in the plasma membrane fraction of the cells, displaying a single class of binding site (E2-dependent: Kd, 32 pM, Bmax, 2.3 pmol/mg protein; E2-independent: Kd, 83 pM, Bmax, 0.26 pmol/mg protein). The ligand binding to both receptors was sensitive to monovalent and divalent cations, and GTP. 2) Among the SRIF analogs tested, the relative potencies of SRIF28 and its analog and cyclosomatostatin compared with SRIF were lower in the E2-dependent receptor than in the E2-independent one. 3) A cross-linking study with N-hydroxysuccinimidyl-4-azido-benzoate revealed that the molecular weight of the cross-linked E2-dependent receptor was approximately 94,000, whereas that of the E2-independent one was 82,000, irrespective of the presence of a reducing reagent. The molecular weight of SRIF receptor from normal male or female rat pituitary was similar to the E2-independent type. 4) Both types of the cross-linked SRIF receptors were solubilized by sucrose monolaurate, adsorbed to a wheat germ agglutinin-agarose column, and eluted with N-acetyl-glucosamine. 5) SRIF inhibited the forskolin-stimulated adenylate cyclase activity in the pituitary membranes from E2-treated rats, but it did not in the E2-depleted membranes. These results demonstrate that there are at least two subtypes of SRIF receptor in the rat anterior pituitary, one of which is exclusively expressed by the treatment with E2, and that these subtypes are distinct with respect to ligand binding specificity, molecular weight, and coupling to adenylate cyclase inhibition.