Orbital cellulitis following cataract surgery.

Orbital cellulitis is an extremely uncommon complication following cataract surgery. Herein, a patient who developed orbital cellulitis less than 24 h after undergoing cataract surgery is described. She responded well to systemic antibiotic treatment and ultimately achieved good visual acuity in the affected eye. The most likely mode of pathogen entry in such cases is considered to be the anaesthetic block given before the cataract surgery. It has been suggested that a careful skin antiseptic preparation before the block is given could prevent this occurrence, but such a preparation did not do so in our case.

Rat milk and dietary long arginine3 insulin-like growth factor I promote intestinal growth of newborn rat pups.

Newborn rat pups were artificially reared by the pup in cup (PIC) method to determine whether dietary long arginine3 IGF-I (long R3 IGF-I), an IGF-I analog with high receptor affinity and low IGF binding protein (IGFBP) affinity, had efficacy on intestinal growth. IGF effects are mediated by IGFBP and receptor interactions, hence dietary-induced changes in intestinal IGF-II receptor patterns and IGFBP-3 message levels were investigated. Intestinal micrographs of pups fed rat milk replacer (RMR) for 3 d showed flattened villi with low cell counts and appeared similar to newborn intestines. Mother-fed (MF) controls and long R3 IGF-I-fed pups showed increased villi height and cell counts when compared with RMR pups, with long R3 IGF-I fed pups showing the greatest increase. At birth IGF-II-specific binding was not uniform in the intestine; specific binding was higher in the proximal intestinal section than in the distal intestinal section. However, after 3 d of MF treatment, specific binding had reversed and the distal section showed higher IGF-II-specific binding. Three days of RMR feeding did not change IGF-II-specific binding from that of the newborn pup. An IGFBP-3 message was identified in intestinal epithelium by in situ hybridization. Northern analysis of IGFBP-3 message showed a decline over time, but the change was not influenced by dietary treatments. In summary, milk-borne growth factors have the potential to affect intestinal growth within 3 d of treatment.

Involvement of prolactin in the REM sleep-promoting activity of systemic vasoactive intestinal peptide (VIP).

The involvement of pituitary prolactin (PRL) in systemic vasoactive intestinal peptide (VIP)-induced sleep was studied. Male rats were implanted with electrodes for EEG-recording, with brain thermistors to record cortical temperature (Tcrt) and with chronic intracardial catheters to obtain blood samples and to deliver substances. One group of rats (n = 8) received normal rabbit serum (NS)+physiological saline (SAL) on the baseline day and was injected with NS+VIP on the experimental day. In the other group of rats (n = 6), the baseline day was followed by administration of PRL-antiserum (PRL-AS) + VIP on the experimental day. The sera and VIP or SAL were injected 30 min before and at light onset, respectively. Sleep-wake activity was then recorded for the next 12-h light period. Systemic VIP-stimulated PRL secretion as measured by RIA in serial samples obtained hour 1 postinjection. VIP also elicited selective increases in REM sleep (REMS) in the rats pretreated with NS. Tcrt was not affected by VIP. Administration of PRL-AS blocked the increase in circulating levels of free (non-IgG-bound) PRL and prevented VIP-enhanced REMS. Comparisons of the sleep effects of PRL-AS+VIP with the previously reported changes in sleep after PRL-AS alone indicate that PRL has a major role in the mediation of the REMS-promoting activity of systemic VIP. The results suggest that an increased release of endogenous pituitary PRL modulates REMS.

Milk-borne factors affect survival, intestinal growth and development in newborn rats.

Early milk of rats contains high concentrations of many hormones and growth factors. Studies were undertaken to evaluate the physiological effects of early rat milk infranatant, insulin-like growth factors (IGFs) and epidermal growth factor (EGF) upon the growth and development of intestinal tissues in newborn rat pups. In one study, pups were artificially reared for 3 days (d2-d5) then fostered back to the dam. Treatment groups were mother fed controls (MF), artificially reared pups fed rat milk replacer (RMR) or RMR with infranatant from rat milk (r-Infra) or bovine milk (b-Infra). After 8 days, survival in the MF and RMR + r-Infra groups was 100% while survival in the RMR + b-Infra and RMR groups were 50 and 30% respectively. In additional experiments, pups were separated from the dam at birth (no suckling) and sacrificed after 2 days of artificial feeding. Treatments included RMR, RMR + r-Infra, and RMR + long Arg-IGF-I + EGF and were compared to MF control. Histological and autoradiographical evaluation of the intestines from MF controls showed advanced differentiation characterized by proliferative cell populations which were restrictively localized to the basal areas of the mucosa. In contrast, intestinal proliferation in the RMR group was diffusely distributed along both the lower regions of the villi and the crypts. Pups fed RMR + r-Infra exhibited intermediate morphology. Pups artificially fed RMR + long Arg-IGF-I + EGF produced a dramatic establishment of an active crypt-villus axis within two days of feeding.(ABSTRACT TRUNCATED AT 250 WORDS)

Hormones and growth factors in milk.

Research dealing with hormones/growth factors in milk has progressed rapidly during the last 10 yr from their identification in milk to their regulation of various functions in the maternal organism and in the neonate. Many hormones, growth factors, and bioactive substances present in the maternal organism are present in colostrum and milk, often exceeding concentrations that occur in maternal plasma. Some appear in milk in different, sometimes multiple, forms from that found in maternal serum, reflecting to some extent synthesis and posttranslational processing by mammary tissue. Recent research has indicated that many milk hormones/growth factors survive the environment of the gut of the neonate, become absorbed into the neonatal circulation, and exert important functions in the neonate.

Bioactive and immunoreactive variants of prolactin in milk and serum of lactating rats and their pups.

Prolactin (PRL)-like bioactivity (in Nb2 lymphoma assay) and immunoreactivity (in radioimmunoassay (RIA)) in rat milk, maternal and neonatal sera and in neonatal rat pituitary cultures were investigated. The PRL-like bioactivity in the water-soluble fraction of rat milk was high and exceeded its immunoreactivity 5.8-, 4.0- and 2.1-fold, on days 2, 12 and 22 of lactation respectively. The elevated bioactivity to immunoreactivity (B/I) ratio of PRL in milk was not due to the presence of interleukin-2 (IL-2) in milk, since the proliferation of the CTLL-2 murine T cells, which are not sensitive to PRL, was promoted by IL-2 but not by milk. Serum levels of immunoreactive PRL were low in sera of non-weaned rat pups on days 2, 12 and 22 postpartum. Similar to milk, the B/I ratio of PRL in sera of rat pups was high and decreased with time postpartum. Pituitary glands of pups obtained on days 2, 12 and 22 secreted progressively increasing amounts of PRL in vitro; the B/I ratio ranged between 1.2 and 2.1 without a significant change. The relative concentrations of size variants in milk were not proportional to those in serum of lactating rats on day 2 postpartum as assessed by Sepharcryl S-100 HR gel permeation chromatography and Nb2 bioassay or RIA. Size variants of biologically active PRL were abundant in early milk and gradually diminished as lactation progressed: a partially resolved peak representing monomeric to dimeric PRL variants (relative molecular weights ranging between 18 k and 42 k) became progressively narrower between days 2 and 22. Biologically active and immunoreactive PRLs displayed disparate elution profiles. The elution profile of PRL in sera of neonatal rats on day 2 post-partum was different from that of maternal serum or milk. The major immunological (and possibly biological) PRL-like activity eluted as two adjacent peaks at 2.2 k and 1.5 k, raising the possibility that fragments of milk-borne PRL were absorbed from the gut after partial proteolytic degradation. In contrast with PRL, GH (which is present in rat milk only in minute concentrations) did not show heterogeneity in sera of 2-day-old rat pups in gel permeation chromatography. The present results demonstrate that the concentrations of PRL-like activity in rat milk and newborn rat serum have been grossly underestimated because levels have been measured by RIA. The high B/I ratio of PRL in milk and neonatal sera is due to the presence of PRL-related compounds.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction between the alpha 2-adrenergic system and nursing in the regulation of growth hormone secretion in the neonatal rat.

Separation of neonatal rats from their mothers decreases, while a subsequent period of suckling (nursing) increases, serum growth hormone (GH) levels in neonatal rats. Milk-borne (humoral) factors and neural factors inherent in mother-offspring interaction have been implicated in these phenomena. Conflicting reports have demonstrated the alpha 2-adrenergic agonist clonidine to increase and to decrease serum GH levels in 10-day-old rats. The present experiments were aimed at testing whether an interaction between the alpha 2-adrenergic system and the nursing-induced changes in GH secretion could account for the discrepancy. Rat pups were treated with clonidine (150 micrograms/kg) or the alpha 2-adrenergic antagonist yohimbine (10 mg/kg), and the drug treatment was combined with separation of the mothers and nursing. Yohimbine did not affect serum GH levels in separated two-day-old pups (i.e. basal levels of the hormone), but prevented the nursing-induced increase in serum GH concentration. In two-day-old pups, clonidine had no effect on basal GH levels but, like yohimbine, prevented the increase in serum GH normally associated with nursing. Both yohimbine and clonidine prevented active sucking behavior, i.e. the pups did not search for and/or attach to the nipples of their mothers. Moreover, the pups treated with yohimbine and clonidine were cooler to the touch than the littermate controls. In eight-day-old pups, yohimbine prevented the nursing-induced increase in serum GH and decreased GH levels below the saline-injected, separated control.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuroendocrine control of immunoreactive growth hormone and bioactive prolactin secretion in neonatal rats: ontogeny and interactions between the serotonergic, cholinergic and alpha 2-adrenergic systems.

The effects of the alpha 2-agonist clonidine (CLO), the serotonin (5-HT) precursor 5-hydroxy-L-tryptophan (5-HTP), the 5-HT2/histamine (H1) antagonist cyproheptadine (CYPRO), the muscarinic cholinergic antagonist atropine (ATR), and an affinity-purified polyclonal anti-rat growth hormone-releasing hormone (rGHRH) immunoglobulin on serum concentrations of growth hormone (GH) and prolactin (PRL) were tested in 2- and 10-day-old litter-mate rat pups. Serum levels of GH and PRL were detected in RIA and Nb2 lymphoma bioassay, respectively. The effects of two different drugs either alone or in combination with each other were evaluated by two-factor analysis of variance. The data indicated that secretion of GH and PRL was regulated by alpha 2-adrenergic, serotonergic and cholinergic mechanisms; the pathways regulating the two hormones, however, were distinct. 5-HTP stimulated GH secretion as early as day 2 postpartum via cholinergic mechanisms not involving GHRH; this pathway was also present in 10-day-old pups. An additional serotonergic pathway was functional in 10-day-old pups which mediated CLO-induced release of GH, and did not include cholinergic transmission.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro control of prolactin (PRL) and growth hormone secretion of neonatal rat pituitary glands: effects of ovine PRL, salmon calcitonin, endothelin-3, angiotensin II, bromocryptine and somatostatin.

Bromocryptine potently decreased prolactin (PRL) secretion of pituitary glands of 2-day-old rats in vitro (up to 85% inhibition; ED50 between 0.1 and 1.0 nM) without altering the bioactivity to immunoreactivity (B/I) ratio. Bromocryptine tended to suppress growth hormone (GH) secretion although the effect did not reach statistical significance. Angiotensin-II (A-II; 1-1000 nM) stimulated PRL secretion in a dose-dependent manner without affecting secretion of GH. The B/I ratio of PRL secreted in response to A-II was increased. Somatostatin (SRIF) had no effect on PRL secretion but inhibited GH secretion in a dose-dependent manner; significant inhibition (50%) was observed at 100 nM. A 6-h exposure to ovine PRL (oPRL) in concentrations equipotent with 1.2-120 ng/ml rat PRL (rPRL) in the Nb2 bioassay had no effect on immunoreactive rPRL secretion. Salmon calcitonin (sCT) and endothelin-3 (ET-3; 0.1-100 nM) failed to inhibit secretion of PRL or GH. PRL secretion was slightly stimulated by sCT with no apparent dose-response relationship. The present findings suggest that neonatal pituitary glands do not display autoregulation of PRL secretion, and sCT and ET-3 (either endogenous or milk-derived) may not function as PRL inhibiting factors in 2-day-old pups. Thus, the receptors of PRL, sCT and ET-3 on lactotropes, or their functional coupling with inhibition of basal PRL secretion, occur at a later stage of development. The specificity of the PRL releasing factor (PRF) activity of A-II at this age is unique for established PRFs and might reflect a physiological function of PRL in osmoregulation. The increased B/I ratio of PRL secreted in response to A-II may be due to the release of specific PRL variants, and might be a sign of functional heterogeneity among lactotropes. The differential sensitivity of PRL and GH to the applied secretagogues suggests that the intracellular regulation of PRL and GH are compartmentalized in the mammosomatotrope cell.

Antiserum to prolactin decreases rapid eye movement sleep (REM sleep) in the male rat.

Previous reports suggest that blood-born prolactin (PRL) may selectively promote rapid eye movement sleep (REMS). To study the possible involvement of endogenous PRL in sleep regulation, rats were systemically injected with either antiserum to PRL or normal rabbit serum, and the sleep-wake activity was determined during the subsequent 12-h light cycle. The administration of normal rabbit serum in physiological saline did not alter sleep-wake activity compared to control recordings, whereas the PRL antiserum caused a modest and selective suppression in REMS. Immunoreactive PRL was eliminated from the serial plasma samples obtained between 6 to 11 h after the injection of the antiserum. Brain temperature was not affected by the antiserum. The results indicate that physiological pituitary PRL secretion has a slight REMS-promoting activity in the male rat. It is speculated that an increased release of pituitary PRL or the PRL-like substance previously demonstrated in the brain may significantly stimulate REMS.

Influence of suckling on tubulin-dependent GTPase activity in the anterior pituitary lobe of the lactating rat.

A GTPase assay was employed to determine the relative proportions of the enzymatic activity in soluble and polymerized tubulin pools in the anterior pituitary lobe of the lactating rat. The GTPase activity in the tubulin fractions was estimated in 25-50 micrograms protein using [gamma-32P]GTP. The liberation of inorganic phosphate (Pi) was proportional to the protein concentration with either of the tubulin fractions. The enzymatic activity appeared to reach equilibrium by 1 min. Antitubulin antibodies inhibited the enzymatic activity in a concentration-dependent manner in both the tubulin fractions; at a final dilution of 1:2000 the antibody maximally inhibited the enzyme activity in both the tubulin fractions by 39-44%. After establishing the optimal conditions for the GTPase assay, the effect of suckling on pituitary GTPase activity was studied. Soluble and polymerized tubulin fractions were prepared from anterior pituitaries obtained from lactating rats killed after suckling for 30, 60, and 90 min; GTPase activity was assayed in both the tubulin fractions in the absence of antitubulin antibody. Compared to the nonsuckled control, suckling for 60 and 90 min stimulated the enzymatic activity in the soluble tubulin fraction by 80% and 44%, respectively (P less than 0.05). The enzymatic activity in the polymerized tubulin fraction increased by 30% at 60 min and decreased by about 20% at 90 min (P less than 0.05). The suckling-stimulated GTPase activity in the two pituitary fractions cannot be attributed to tubulin alone since there are other proteins also capable of hydrolyzing GTP. Therefore, GTPase activity was assayed in the pituitary tubulin fractions in the presence of antitubulin antibody (1:2000 dilution); tubulin-GTPase activity is the difference between the activity assayed in the absence of the antibody and that which was determined in the presence of the antibody. In the soluble tubulin fraction, tubulin-GTPase activity increased by 166% at 30 min suckling (P less than 0.05), decreased by 40% at 60 min (P less than 0.05), and again increased by 148% at 90 min (P less than 0.05). In the polymerized tubulin fraction, the enzyme activity decreased by 82% at 30 min (P less than 0.05), increased by 742% at 60 min (P less than 0.05), and again decreased by 95% at 90 min (P less than 0.05). Thus, an inverse relationship between tubulin-GTPase activities in the two pituitary fractions was observed and provides further evidence in support of our hypothesis that microtubules are recruited to transport PRL granules from the Golgi apparatus to the plasma membrane.

Regulation of prolactin transformation in the rat pituitary.

We have investigated the mechanisms involved in the inhibition by dopamine of the transformation of prolactin within the anterior pituitary of the lactating rat. The degree of inhibition depends on the intracellular age of prolactin, being greater in newly synthesized (<1 hour) and in older (>12 hours since biosynthesis) hormone and lesser in prolactin synthesized 4-8 hours earlier. Transformation occurs in prolactin granules and involves an increase in oligomeric forms of prolactin at the expense of the monomeric form. A reversible disulphide-linked mechanism may be involved in dopamine inhibition of prolactin transformation; it is dependent upon the intracellular and/or intragranular pH via a Na+/H+ exchange mechanism. Transient suppression of dopamine inhibition may lower the intracellular/intragranular pH and subsequently cause transformation of the hormone. Developmentally, dopamine secretion by tuberoinfundibular neurons is seriously impaired and the response of pituitary lactotrophs to dopamine is reduced in adult rats deprived of milk prolactin during Days 2-5 post partum. These results suggest milk prolactin ingested during a critical post partum period may exert an organizational effect upon dopamine secretion and its function on the pituitary lactotroph during adulthood.

Thyrotropin-releasing hormone mediates growth hormone release induced by milk and nursing in neonatal rats.

Previous evidence from this laboratory suggested that growth hormone (GH) release induced by milk in vitro and by nursing in vivo from neonatal rat pituitary glands is mediated by an alternative GH-releasing factor(s) (GRF) distinct from GH-releasing hormone (GHRH(1-43) ). In the present experiments we tested whether thyrotropin-releasing hormone (TRH) could fulfil the criteria of this alternative GRF in neonatal rats. The water-soluble fraction of rat milk (infranatant, prepared by ultracentrifugation) and its methanol/acetic acid extract (milk-borne peptides) stimulated GH release from perifused pituitary glands obtained from 2-day-old rats. Dialysis of the infranatant (mol wt cut-off: 2,000) against 500 volumes of culture medium at 4°C eliminated its GH-releasing activity in the perifusion system, while the infranatant retained its full GRF-like activity when incubated at 4°C without dialysis. The milk-borne GRF eluted as a single peak and coeluted with TRH in a combined gel permeation chromatography (Sephadex G-10) and perifusion set-up. Prolactin secretion was also stimulated simultaneously with the release of GH induced either by milk or by TRH. In a stepwise C(18) reversed-phase chromatography, milk-borne GRF was highly hydrophilic and coeluted with synthetic TRH. The in vitro GH-releasing bioactivities of synthetic TRH and a milk extract purified in C(18) reversed-phase chromatography were abolished by proline-specific endopeptidase. Thus, TRH and milk-borne GRF displayed similar molecular weights, hydropathic characteristics and proteolytic enzyme resistance. In vivo, nursing (which has been reported as a potent stimulus of GH secretion even in the absence of milk-intake) increased serum GH levels in 2-day-old pups. A supramaximal dose of TRH (10 ng/g intraperitoneally) stimulated GH release in 2-day-old pups separated from their mothers for 6 h to a similar extent as nursing. Nursing-induced levels of serum GH were not further elevated by TRH. This failure of TRH to further increase serum GH levels was not due to a maximal GH output by the neonatal pituitary gland, since the GH release induced by the serotonin precursor 5-hydroxy-L-tryptophan was augmented either by TRH or by nursing. These data provide evidence that the milk-borne GRF-like activity in vitro is indistinguishable from TRH, and suggest that TRH (probably of hypothalamic origin) might be the mediator of the nursing-induced release of GH in vivo as a physiological GRF in neonatal rats.

Rat milk inhibits the mitogenic response of Nb2 lymphoma cells to prolactin.

Prolactin-like bioactivity in the rat milk was observed using the lactogen specific Nb2 lymphoma assay. The water soluble fraction (infranatant) of pooled milk samples obtained on days 2, 12 or 22 postpartum stimulated Nb2 cell growth in the range of 0.08-2.5 microliters/well. Higher concentrations of day-12 and day-22 (but not day-2) milk infranatant, however, decreased Nb2 proliferation in a dose-dependent fashion. [3H]-thymidine incorporation used as an indicator of cell growth was decreased by 21%, 49% and 83% at the doses of 5, 10, 20 microliters/well concentrations of day-22 milk infranatant, respectively. Milk infranatant did not reduce cell viability as assessed by Erythrosin B exclusion test. Addition of exogenous rat PRL (NIH B-6) at concentrations of ED50-ED90 did not restore the Nb2 proliferation rate decreased by milk infranatant. Saturating doses of PRL (ED100-ED400) resulted in maximal cell growth, but failed to counteract the inhibitory effect of milk infranatant. The relative molecular weight of the putative Nb2 cell inhibitor of rat milk is between 10 kDa and 30 kDa as determined by ultrafiltration and dialysis. The inhibitory activity of milk infranatant is stable at physiological pH, but is destroyed upon acidification. Thirty min of incubation at 37 degrees C enhanced but 30 min of incubation at 100 degrees C only slightly decreased the calculated total inhibitory effect of milk infranatant. These initial results indicate the presence of a water-soluble antimitogenic factor in rat milk (rMAF) which inhibits the Nb2 lymphoma cell response to prolactin in a non-competitive manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular heterogeneity of prolactin in lactating rats and their pups: biological and immunological activities in pituitary gland, serum and milk.

Prolactin (PRL)-like bioactivities (in Nb2 lymphoma assay), immunoreactivities (in RIA) and B/I ratios in rat milk, maternal and neonatal pituitary glands and sera were investigated. The PRL-like bioactivity in the water-soluble fraction of rat milk (infranatant prepared by ultracentrifugation) exceeded its immunoreactivity 3-7-fold. The elevated B/I ratio was in part due to the presence of a glycosalated PRL (G-rPRL)-like material, since 5-70% of the PRL-like bioactivity was recovered from the glycosylated fraction of rat milk infranatant prepared by concanavalin-A affinity chromatography. We were unable to detect PRL-like immunoreactivity in the glycosylated fraction of rat milk, and calculated that the maximal cross-reactivity of G-rPRL in the RIA is less than 3.8%. In day 12 milk, over 80% of the G-rPRL-like bioactivity eluted from a Sephadex G-100 column as a high apparent molecular weight (Mr) substance (approximately 50 kD), while the rest eluted as a monomeric G-rPRL (24-25 kD). The PRL-like bioactivity in the nonglycosylated fraction eluted in three peaks (Mr: 50, 24 and 16 kD), while two immunoreactive peaks occurred (Mr: 24 and 8 kD). The concentration of rPRL-like immunoreactivity in rat milk increased during the first days of lactation, remained high in midlactation, and declined by the end of lactation. The PRL-like bioactivity in the nonglycosylated fraction of rat milk displayed a similar timecourse. G-rPRL-like bioactivity in rat milk, however, changes inversely, i.e. decreased between days 2 and 18 postpartum then increased by day 22. The concentration of high Mr PRL-like bioactivity in rat milk was greatly reduced by day 22 from day 2 postpartum. No PRL-like bioactivity or immunoreactivity was recovered from the IgG fraction (prepared by protein A affinity chromatography) of rat milk. The B/I ratio in day 2 maternal pituitary glands was close to 1. In neonatal pituitaries and in maternal sera, however, the B/I ratio was slightly elevated (2-3). The B/I ratio in day 2 neonatal serum was between 6 and 22, while the B/I ratio of PRL secreted by day 2 neonatal pituitary glands in vitro was 1. The present results demonstrate that the concentrations of PRL in rat milk and neonatal serum have been grossly underestimated because levels were detected by RIA. The high B/I ratio reflects the presence of PRL variants. Milk appears to be the most likely source of PRL variants in the circulation of the neonate.(ABSTRACT TRUNCATED AT 400 WORDS)

Prolactin stimulates the release of oxytocin in lactating rats: evidence for a physiological role via an action at the neural lobe.

The present studies were designed to investigate whether prolactin (PRL) influences the secretion of oxytocin (OT) in lactating rats, and to test whether the previously reported inhibitory and stimulatory effects of dopamine-2 (D-2) agonists and antagonists, respectively, on OT release might be secondary to their respective inhibitory and stimulatory effects on the release of PRL. Intravenous administration of either rat (r) or ovine (o) PRL to lactating, nonsuckled rats increased basal plasma concentrations of OT. rGH was ineffective, but administration of oGH did produce some stimulation of OT release. Both oPRL and rPRL significantly enhanced the electrical stimulation-induced release of OT from isolated stalk-neurointermediate lobes, in vitro, without affecting the basal release of the peptide. oGH was ineffective on basal or stimulated in vitro OT release, and neither hormone altered basal or stimulation-induced release of vasopressin from these tissues. Both rPRL and oPRL reversed the inhibitory effect of the D-2 dopamine agonist bromocriptine. Immunoneutralization of circulating PRL with a highly specific antiserum abolished the increases in OT in response to either suckling or to administration of the D-2 dopamine antagonist domperidone.(ABSTRACT TRUNCATED AT 250 WORDS)

Excitatory and inhibitory dopaminergic regulation of oxytocin secretion in the lactating rat: evidence for respective mediation by D-1 and D-2 dopamine receptor subtypes.

The present experiments were designed to test whether the previously reported excitatory and inhibitory effects of dopamine (DA) on the secretion of oxytocin (OT) in lactating rats are exerted at different DA receptor subtypes, and to examine whether one or both of these effects might occur at the level of the posterior pituitary. The basal release of OT in nonsuckled, lactating rats was increased after intravenous administration of the D-1 DA agonist SKF 38393, and this effect, as well as the suckling-induced release of OT, was prevented by treatment with the D-1 DA antagonist SCH 23390, suggesting that DA may exert an important stimulatory influence over OT secretion through an action at the D-1 DA receptor subtype. A small stimulation of basal PRL release was also produced by SKF 38393, but blockade of the D-1 DA receptor did not prevent the suckling-induced release of this hormone. Stimulation of the D-2 DA receptor with PPHT had no effect on basal OT release in nonsuckled rats, but this agent, as well as another D-2 DA agonist, bromocriptine, prevented the suckling-induced release of both OT and PRL. The inhibitory effect of D-2 DA receptor stimulation was blocked by the D-2 DA antagonist domperidone, which increased the basal release of both hormones when given alone. These observations confirm previous findings that inhibitory effects of DA on suckling-induced OT release are mediated through activation of the D-2 DA receptor. To test whether either dopaminergic effect occurs at the level of neurosecretory endings in the neurointermediate lobe (NIL), the stalk-NIL was isolated from lactating rats and perifused in vitro. The stalk-NIL junction was electrically stimulated for 4 s, and the effects of selective D-1 DA and D-2 DA agonists and antagonists on the basal and electrically evoked release of OT and vasopressin (VP) was assessed using the two stimulation (S2/S1) paradigm. Electrical stimulation produced marked increases in release of both neural lobe peptides in a Ca(2+)-dependent manner, and the electrically evoked release of OT, but not VP, was enhanced by the opiate antagonist naltrexone (10 microM). Consistent with the in vivo results, SKF-38393 (20 microM) produced a small, but statistically significant, increase in electrically induced OT release, while SCH 23390 (20 microM) was without significant effect. Neither drug affected the basal release of OT or the basal or electrically stimulated release of VP.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of Basal and nursing-induced secretion of growth hormone in the neonatal rat: the involvement of serotonergic, muscarinic cholinergic, alpha-adrenergic, somatostatin and growth hormone-releasing hormone systems.

Abstract Various neural factors are involved in the suckling-induced increase in serum growth hormone (GH) levels in neonatal rats, and, in the present study the serotonergic, cholinergic, somatostatin and GH-releasing hormone (GHRH) systems were investigated. The serotonin (5-HT) precursor 5-hydroxy-L-tryptophan (5-HTP) and the 5-HT receptor agonist quipazine maleate stimulated serum GH levels in 2-day-old rat pups separated from their mothers for 6 h. The increase in serum GH during suckling was further elevated by 5-HTP. The 5-HT antagonist cyproheptadine decreased serum GH levels in separated 2-day-old pups, and although it reduced the amplitude of the suckling-induced increase in serum GH concentration, it did not alter the increase in serum GH on a percentage basis. The effect of the cholinergic muscarinic antagonist atropine sulfate (ATR) was similar to that of cyproheptadine. Moreover, in separated pups, ATR prevented the increase in serum GH induced by 5-HTP. In contrast with 2-day-old pups, ATR completely eliminated the suckling-induced release of GH in 10-day-old rats. However, ATR failed to prevent GH release induced by the alpha(2)-adrenergic agonist clonidine HCI in 10-day-old male pups. While thyrotropin-releasing hormone increased serum GH levels, rat GHRH failed to alter serum GH levels either in separated or in suckled 2-day-old rat pups. Immunoneutralization for rat GHRH eliminated the increase in serum GH induced by clonidine HCI in 10-day-old pups, but (on a percentage basis) failed to prevent the GH-increasing effect of suckling in 2-day-old pups. While somatostatin failed to significantly decrease serum GH in separated 2-day-old pups, it effectively decreased serum GH levels in 2-day-old pups which were suckled. Cysteamine, which depletes hypothalamic somatostatin, increased serum GH in separated 2-day-old pups, and further increased the suckling-induced levels of serum GH. Cysteamine partially prevented the GH-decreasing effect of ATR. The present findings suggest that 1) the serotonergic and cholinergic systems are involved in the regulation of GH secretion as early as day 2 postpartum; 2) the serotonergic and cholinergic systems modulate the basal, and do not modulate the suckling-induced levels of serum GH; 3) the serotonergic system may exert its stimulatory influence on GH secretion only in the presence of a functional muscarinic cholinergic system; 4) the cholinergic system, at least in part, stimulates GH secretion via a cysteamine-sensitive system (probably by inhibiting somatostatin); 5) the cholinergic system is not functionally coupled with the alpha(2)-adrenergic system, which stimulates GH secretion via rat GHRH; 6) since in 10-day-old pups clonidine HCI was effective only in males, while suckling was effective in both sexes, the alpha(2)-adrenergic system is not involved in the suckling-induced increase of serum GH; and finally 7) neither somatostatin nor rat GHRH seem to be involved in the suckling-induced changes in serum GH. The findings are consistent with the hypothesis that the high circulating GH levels in the neonatal rat are due to alternative GH-releasing factors, perhaps thyrotropin-releasing hormone or gamma-aminobutyric acid. The neurohumoral mediator of the suckling-induced GH release in neonatal rats remains to be identified.

Effect of ovine prolactin on tubulin function in the anterior pituitary lobe of the lactating rat.

Ovine PRL (oPRL) was employed to investigate the role of anterior pituitary lobe microtubules in suckling-induced PRL secretion in the lactating rat. Groups of primiparous rats on days 12-14 postpartum were isolated from their pups for 4-5 h, then suckled for 10 min, and killed, and the anterior pituitary lobes were dissected out. Each pituitary lobe was processed to obtain the two tubulin pools, viz. soluble and polymerized tubulin fractions. After 10 min of suckling the pituitary soluble tubulin levels were reduced by about 25% (P less than 0.05), and polymerized tubulin levels increased by about 40% (P less than 0.05). When 3 mg oPRL were injected 4 h before suckling, the suckling-induced rise in the polymerized tubulin levels in the anterior pituitary lobe as well as plasma PRL levels were significantly inhibited (P less than 0.05). In the suckled rats injected with oPRL a 25% reduction in the total tubulin levels (soluble and polymerized) was observed. In a second experiment, each anterior pituitary lobe obtained from groups of rats suckled for 10 min was processed to obtain the total tubulin fraction. Suckling for 10 min stimulated the in vitro assembly of total tubulin fraction into microtubules by about 150% (P less than 0.05); 3 mg oPRL injected 4 h before suckling inhibited the suckling-induced rise in tubulin assembly (P less than 0.05). In a third experiment, suckling for 10 min stimulated GTPase activity in the total tubulin fraction by about 60% (P less than 0.05). Administration of oPRL 4 h before suckling caused about an 80% increase in GTPase activity. At a 1:2000 dilution, antitubulin antibodies maximally inhibited GTPase activity by about 40%, suggesting that a significant proportion of the enzyme activity can be due to tubulin present in the pituitary. These results suggest that PRL secretion is coupled to pituitary microtubules and that in addition to tubulin, other GTP-binding proteins might be involved in PRL secretion.