Combined strategies following surgical drainage for perianal fistulizing Crohn's disease: failure rates and prognostic factors.

AIM: The medico-surgical strategy for the treatment of perianal fistulizing Crohn's disease (CD) following surgical drainage remains challenging and debated. Our aims were to describe the failure rate of therapeutic interventions after drainage of the fistula tract and determine the factors associated with failure to optimize medico-surgical strategies. METHOD: All consecutive patients with perianal fistulizing CD who underwent surgical drainage with at least a 12-week follow-up were included. Failure was defined as the occurrence of at least one of the following items: abscess recurrence, purulent discharge from the tract, visible external opening and further drainage procedure(s). RESULTS: One hundred and sixty-nine patients were included. The median follow-up was 4.0 years. The cumulative failure rates were 20%, 30% and 36% at 1, 3 and 5 years, respectively. The cumulative failure rates in patients who had sphincter-sparing surgeries or seton removal were significantly higher than in those who had a fistulotomy. Anterior fistula [hazard ratio (HR) = 2.52 (1.13-5.61), P = 0.024], supralevator extension [HR = 20.78 (3.38-127.80), P = 0.001] and the absence or discontinuation of immunosuppressants after anal drainage [HR = 3.74 (1.11-12.5), P = 0.032] were significantly associated with failure in the multivariate analysis model. CONCLUSION: Combined strategies for perianal fistulizing CD lead to a failure rate of 36% at 5 years. Where advisable, fistulotomy may be preferred because it has a lower rate of recurrence. The benefits of immunosuppressants require a dedicated prospective randomized trial.

Sphincter-sparing surgery for complex anal fistulas: radiofrequency thermocoagulation of the tract is of no help.

AIM: To compare the rate of failure of radiofrequency thermocoagulation for anal fistula with that of rectal advancement flap in a case-matched study. METHOD: Patients who underwent radiofrequency treatment were compared with age- and sex-matched patients with Crohn's disease (CD) who underwent a rectal flap procedure. Fistula features, general characteristics and the main clinical events were recorded in a prospective database. Failure was defined by at least one of following: abscess, purulent discharge, visible external opening or further drainage procedure. RESULTS: A total of 62 patients [median age 45 (range 36.8-57.5) years; 22 women, 40 men; 22 with CD] were analysed. The failure rate of radiofrequency treatment was higher than that of rectal flap treatment (74.2% vs 32.2%; P = 0.004). The cumulative probabilities of failure of the radiofrequency treatment were 53.8% (38.8-68.3), 71.8% (55.3-84.0) and 87.4% (70.6-95.3) at 3, 6 and 12 months, respectively. Three patients in the radiofrequency group required drainage for an abscess and one had severe thermal ulceration. The Cox proportional hazards regression model (surgical procedure, obesity, CD) showed rectal flap treatment [3.48 (1.60-8.07); P = 0.001] and CD [2.60 (1.16-6.41); P = 0.02] to be the main independent predictors of healing. CONCLUSION: Radiofrequency thermocoagulation is a less satisfactory sphincter-sparing treatment for the management of anal fistula than a rectal flap procedure.

Advancement flap procedure in Crohn and non-Crohn perineal fistulas: a simple surgical approach.

AIM: Rectal flap advancement is still a part of therapeutic management of anal fistulas. Data on the outcome of rectal flap advancement in patients with Crohn's disease (CD) is scarce. Our objective was to ascertain rates of failure of rectal flap advancement and to determine predictive factors for failure, with a special focus on CD METHOD: The patients' details, the characteristics of the fistula and the main clinical and therapeutic events were prospectively assessed among patients who underwent rectal flap advancement. All patients had a partial-thickness rectal flap advancement. Failure of primary rectal flap advancement was defined as the occurrence of at least one of the following: abscess, discharge, visible external opening, further drainage procedure. The rates of failure of rectal flap and the predictive factors of failure were assessed. RESULTS: Eighty-seven patients (34 patients with CD) were included. The median (interquartile range) follow-up was 13.3 (3.8-38.1) months. The cumulative failure rates were 15.9% (10.3-23.6), 23.0% (16.0-31.8), 31.6% (22.9-41.8) and 41.3% (30.5-53.0) at 3, 6, 12 and 24 months respectively. These data were comparable in Crohn's patients. Those with a supralevator fistula [hazard ratio 2.53 (1.01-7.71), P = 0.0476] and patients who had fewer than two fistula drainages before rectal flap [hazard ratio 3.19 (1.40-8.23), P = 0.005] were associated with higher rectal flap failure rates. In CD patients, the absence of biological therapy at referral was predictive of failure. CONCLUSION: Rectal flap advancement is a satisfactory option for the therapeutic management of anal fistula, including CD populations. Fistula drainage is needed before performing this surgical technique.

Nonlinear analysis of orthostatic posture in patients with vertigo or balance disorders.

The stabilometry signals involve irregular and unpredictable components. In order to identify the hidden dynamics that underlie the multi-link networks consisted of the multiple sensory systems, motor components and central integration, we applied a nonlinear analysis to these signals. We evaluated the postural control differences between eyes open and closed by means of the dynamical closeness between two states, known as similarity index, for the patients with vestibular disorders. We were able to demonstrate that some patients (eight of 21) showed a difference between the conventional and nonlinear measures. Especially, the similarity index tended to reflect the clinical course of the vestibular compensation and the findings in the patients with benign paroxysmal positional vertigo (BPPV) demonstrated that its vestibular function may include various pathologies besides canalithiasis. These results suggest that nonlinear analysis can elucidate the complex postural control networks and this procedure may also be able to provide the new findings of the stabilometry examinations.

Involvement of sst2 somatostatin receptor in locomotor, exploratory activity and emotional reactivity in mice.

Somatostatin (SRIF) controls many physiological and pathological processes in the central nervous system but the respective roles of the five receptor isotypes (sst1-5) that mediate its effects are yet to be defined. In the present study, we attempted to identify functions of the sst2 receptor using mice with no functional copy of this gene (sst2 KO mice). In contrast with control 129Sv/C57Bl6 mice, sst2 mRNA was no longer detectable in the brain of sst2 KO mice; 125I-labeled Tyr0DTrp8-SRIF14 binding was also greatly reduced in almost all brain structures except for the hippocampal CA1 area, demonstrating that sst2 accounts for most SRIF binding in mouse brain. Invalidation of this subtype generated an increased anxiety-related behaviour in a number of behavioural paradigms, while locomotor and exploratory activity was decreased in stress-inducing situations. No major motor defects could be detected. sst2 KO mice also displayed increased release of pituitary ACTH, a main regulator of the stress response. Thus, somatostatin, via sst2 receptor isotype pathways, appears involved in the modulation of locomotor, exploratory and emotional reactivity in mice.

Delta opiate receptors account for the castration-induced unmasking of gonadotropin-releasing hormone binding sites in the rat pituitary.

Under control incubation conditions, gonadotropin-releasing hormone (GnRH) binds only a fraction of its receptors in rat-cultivated pituitary cells. Unmasking of the remaining receptors, which have been termed 'cryptic', requires drug- or peptide-induced protein kinase activation. Spontaneous masking however is not observed on pituitary cells sampled from castrated male rats, suggesting the presence of an intrinsic unmasking factor. Many endogenous factors could theoretically account for the effect. Here we attempted to identify the factor involved by taking advantage of their differential dependency upon second messengers and transduction cascades. Spontaneous unmasking of GnRH binding was found reversed by pertussis toxin (PTX), an inhibitor of alphai and alphao subunits of heterotrimeric G proteins, and by U73122, a phospholipase C (PLC) inhibitor. In contrast, desensitization of protein kinase C (PKC) or inhibition of tyrosine kinase by herbimycin were ineffective. Among endogenous pituitary factors able to unmask GnRH receptors in pituitary cells from normal male rats, as EGF, NPY or opiate peptides, only the latter were found to correspond to this transduction profile. In an attempt to characterize the pharmacology of opiate effects, naloxone (10 microM), a poorly selective opiate antagonist, restored masking of GnRH binding in cells from castrates. Only the delta antagonist naltrindole (1 microM) was able to mimick the action of naloxone. Conversely, when tested on cells from intact animals, morphine (10 microM), as well as dslet (1 microM) and met-ENK (10 nM), preferential delta agonists, but not dago and beta-endorphin or U50488 H and dynorphin, respectively micro and kappa agonists, were able to suppress masking. Among opioid peptides endogenous to the pituitary, only met-ENK was able to unmask cryptic receptors, an effect antagonized by naltrindole. We conclude that an opiate delta receptor subtype is endogenously activated in the pituitary of castrated male rats to prevent masking of GnRH binding.

Cryptic gonadotropin-releasing hormone receptors of rat pituitary cells in culture are unmasked by epidermal growth factor.

Protein kinase activators as well as several neuropeptides are able to increase the GnRH-binding capacity of cultured adenohypophyseal cells. To determine whether such up-regulation of GnRH-binding sites can be achieved by a substance(s) endogenous to the pituitary, binding experiments were performed after exposure of cells to increasing amounts of medium conditioned by incubation with primary cultures of adenohypophyseal cells for 4 days. Addition of the conditioned medium elicited a 50% increase in GnRH binding. Characterization of the agent(s) responsible for the effect was attempted by submitting the conditioned medium to molecular sieve filtration, adding or immunoprecipitating endogenous substances, and comparing the susceptibilities of the responses to various inhibitors of transduction processes. Fractionation of the medium indicated that active molecules were of a proteic nature, with M(r) ranging from 5,000-10,000. Among major endogenous moieties corresponding to these criteria [epidermal] growth factor (EGF), transforming growth factor-alpha, and insulin-like growth factors I and II), only the first two exhibited properties similar to those of the conditioned medium. EGF stimulated binding with an EC50 of 3.6 +/- 0.8 pM. Immunoprecipitation of EGF, but not transforming growth factor-alpha, inactivated the conditioned medium. The effects of both conditioned medium and EGF were inhibited by herbimycin, a tyrosine kinase inhibitor; U73122, a phospholipase C inhibitor; and prior desensitization of protein kinase C. In contrast, both were insensitive to pertussis toxin pretreatment. In parallel, EGF did not increase LH secretion by itself, but potentiated its response to GnRH in a concentration range of 1 pM to 1 nM, resulting in a shift of the curve toward lower values of GnRH. It is concluded that EGF is able to control the accessibility of binding sites to GnRH and to potentiate the responsiveness of gonadotropes to the decapeptide.

Rho proteins are localized with different membrane compartments involved in vesicular trafficking in anterior pituitary cells.

In order to explore the role of certain GTP binding proteins in the rat anterior pituitary, we have analyzed the subcellular distribution of the proteins rho and rab. They were found in both membrane and cytosolic fractions. Rab1 and rab2 were localized in both Golgi and endoplasmic reticulum (ER) membranes, while rab4 and rab6 were found in fractions enriched with Golgi and plasma membranes, implicating these proteins in the control of vesicular intracellular trafficking as described in other systems. Rab3 was localized like a fraction of synaptophysin, suggesting a role for rab3 in the targeting of "synaptic-like' microvesicles. We have identified three substrates of C. botulinum exoenzyme C3. A 26-kDa substrate with an isoelectric point (pI) of 5.2, probably rhoB, was localized in the lightest fractions such as rab3 and synaptophysin proteins. Two other 23-24 kDa substrates with pI of 5.5-5.8, probably rhoA and/or rhoC, were found in both fractions enriched with ER and secretory granules. Rho proteins have been implicated in the control of actin polymerization. Their localization in anterior pituitary suggests that rhoB could control the association of synaptic-like microvesicles and plasma membrane, and that rhoA/rhoC could play a role in secretory granule exocytosis; these two pathways being involved in cytoskeleton protein reorganisation in response to extracellular signals.

Neuropeptide Y enhances LHRH binding to rat gonadotrophs in primary culture.

Incubation of dispersed adenohypophyseal cells from intact male rats with Neuropeptide Y (NPY) or Peptide YY (YY) at 21 degrees C increased maximal 125I LHRHa binding (Bmax) by about 50%. In presence of 10(-7) M NPY, Bmax calculated from saturation isotherm curves was 15.3 +/- 1.9 fmoles x mg-1 proteins, as compared to 10 +/- 1 fmoles x mg-1 in control incubates. The increase was dose dependent with an EC50 of 6.3 +/- 1.8 10(-10) M NPY. Preincubation of the cells with pertussis toxin (PT, 15 ng/ml) for 24 h abolished the effect, suggesting coupling of NPY receptors to G alpha o or G alpha i proteins. NPY 10(-7) M inhibited basal and Forskolin 10(-5) M stimulated intracellular cyclic AMP formation by 31.9 +/- 3.4% and 30.6 +/- 2.3% respectively. Desensitization of protein kinase C by overnight preincubation of the cells with 10(-6) M phorbol ester (PMA) did not interfere with the effect of NPY. In contrast, W7, a calmodulin inhibitor, as well as H7, a protein kinase C inhibitor with a relatively wide spectrum, suppressed the effect of NPY with IC50 of 1.4 +/- 0.6 10(-6) M and 2.2 +/- 0.5 10(-5) M, respectively. Taken together, these results suggest that NPY is able to control unmasking of a cryptic LHRH receptor pool in pituitary cells by a process dependent upon both GTP binding proteins and calmodulin dependent protein kinase.

Preferential distribution of C-terminal fragments of [hydroxyproline9]LHRH in the rat hippocampus and olfactory bulb.

Several molecular forms related to the decapeptide LHRH were characterized and quantified in various brain structures of intact and castrated male and female rats. Distinct moieties were separated by high performance liquid chromatography (HPLC) and radioimmunoassayed against anti-LHRH antibodies of different specificities. The hypothalamus contained the highest concentration of LHRH-like material detected by the antisera. The predominant (89%) molecular form recovered from that structure was LHRH itself; 9% of the material corresponded to [hydroxyproline9]LHRH ([Hyp9]LHRH), an endogenous posttranslational product of the LHRH precursor, and the residual immunoreactivity was accounted for by C-terminal fragments of both decapeptides, as assessed after labelling HPLC columns with appropriate synthetic or endogenous hypothalamic peptides. The proportions were the same in both sexes and were not affected by castration, in spite of a lesser overall LHRH activity in females and in castrates. LHRH and [Hyp9]LHRH were also detected in the olfactory bulb and the hippocampus. In these structures however, most (97%) LHRH-related molecules corresponded to C-fragments derived from [Hyp9]LHRH, whereas only very few fragments derived from the nonhydroxylated decapeptide were found. Sex or castration affected neither total nor relative concentrations of LHRH-derived molecules in the olfactory bulb and the hippocampus. Taken altogether, these observations are suggestive of a different LHRH metabolic regulation in neurons projecting to either the median eminence or extrahypothalamic areas. In the latter case, larger amounts of the LHRH precursor appear processed to [Hyp9]LHRH. Recovery of relatively high concentrations of [Hyp9]LHRH C-fragments in the olfactory bulb and the hippocampus reflects the higher resistance of the Hyp9-Gly10-NH2 than the Pro9-Gly10-NH2 peptide bond to hydrolysis by the postproline cleaving enzyme. In view of reports that intracerebral administration of C-terminal fragments of LHRH are able to trigger sex behavior, our finding that extrahypothalamic structures contain relatively high concentrations of the [Hyp9]LHRH-derived, more stable C-fragments suggests that these catabolites may have a role in the regulation of sex behavior.

A second endogenous molecular form of mammalian hypothalamic luteinizing hormone-releasing hormone (LHRH), (hydroxyproline9)LHRH, releases luteinizing hormone and follicle-stimulating hormone in vitro and in vivo.

In vitro and in vivo release of pituitary hormones were studied in the presence of (hydroxyproline9)LHRH ((Hyp)LHRH), a newly characterized endogenous molecular form of LHRH. Results were compared to those obtained with LHRH itself. (Hyp)LHRH, as LHRH, stimulated both luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release in a homothetic manner. The hydroxylated compound was, however, 24 times (in vitro) and 5 times (in vivo) less potent than LHRH. The lower activity of (Hyp)LHRH than of LHRH in the in vitro assay correlated well with a 28-fold lesser potency in a binding test using pituitary membrane preparations. The higher relative potency and the prolonged effect of (Hyp)LHRH in the in vivo test were related to a lesser susceptibility of the hydroxylated form to proteolytic degradation. Effects of LHRH and of (Hyp)LHRH were not additive, both peptides were equally able to desensitize gonadotrophs to a subsequent challenge by the other. Taken together, these observations suggest that both forms of LHRH act at the same receptor site. The lesser affinity of the hydroxylated compound is compensated to a certain extent by its higher resistance to enzymatic degradation. It is concluded that in spite of its lesser potency, (Hyp)LHRH may participate in the regulation of gonadotropins.

A Ca2+ calmodulin dependent kinase rather than protein kinase C is involved in up-regulation of the LHRH receptor.

Stimulation of protein kinase C (PKC) by phorbol ester (PMA) was reported previously to increase total binding of the peptide in whole rat pituitary cells. The effect could be obtained in cells from intact, not from spayed animals, suggesting a different level of spontaneous phosphorylation in both conditions. In the present work, endogenous PKC was desensitized in pituitary cells sampled from intact or 3 weeks castrated male rats and maintained in primary culture. Desensitization was induced by overnight incubation with 1 microM PMA. The maximum number of plasma membrane LHRH receptors (Bmax) present on cells from in intact animals was higher (+ 98 +/- 9%) when binding was performed at 0.5 degrees C instead of 21 degrees C as already observed in non PKC-desensitized cells. PMA (100 nM) was ineffective to increase Bmax, suggesting effectiveness of enzyme desensitization. In contrast, ionomycin 1 microM increased Bmax (53 +/- 10%). This increment was inhibited by W7, a calmodulin inhibitor, with an IC50 = 1 +/- 0.35 10(-6) M. No temperature dependency of the Bmax was observed in cells from castrated rats as already shown in the absence of PKC desensitization. Under these conditions, a Bmax decrease of 34 +/- 6% and 36.5 +/- 7.5% respectively was observed in the presence of H7, a PKC inhibitor, or of W7 (IC50 = 1 +/- 0.5 10(-5) M and IC50 = 0.8 +/- 0.2 10(-6) M). We conclude that a Ca2+ calmodulin dependent protein kinase rather than PKC itself is responsible for unmasking LHRH receptors.

[New data on a second endogenous molecular form of mammalian hypothalamic LHRH, (hydroxyproline 9) LHRH]. / Nouvelles données sur une seconde forme moléculaire endogène de la LHRH hypothalamique de mammifère, la (hydroxyproline9) LHRH.

An endogenous hydroxylated form of LHRH, (Hyp) LHRH, is able to displace LHRH bound to pituitary membrane preparations. In parallel, it stimulates release of both LH and FSH from pituitary cells in primary culture. The potency ratio of (Hyp)LHRH is approximately 1:20 and 1:5 with respect to the native decapeptide when peptidasic degradation is or is not inhibited. This correlates with a greater resistance of (Hyp) LHRH towards enzymatic degradation; in contrast to LHRH, the C-terminal (residues 6 to 10) end of (Hyp) LHRH is not degraded and generates C-terminal fragments which account for 64% of the LHRH immunoreactivity in extrahypothalamic areas as the hippocampus. Besides its weak gonadotropin releasing activity and its action or its localization in peripheral organs (placenta, gonads), a major role of the hydroxylated decapeptide may thus be to serve as a precursor of smaller active fragments on targets other than pituitary receptors.

Characterization and distribution of receptors for gonadotropin-releasing hormone in the rat hippocampus.

Distribution and properties of receptors for gonadotropin-releasing hormone (GnRH) were analyzed in the brain of adult male rats. Binding of the iodinated GnRH agonist Des-Gly10-(D-Ala6)-GnRH ethylamide was studied in hippocampus and anterior pituitary using three convergent approaches: quantitative autoradiography on frozen tissue, binding to fresh slices, and binding to crude membrane preparations. In all cases, binding was specific, saturable, and time, pH, and temperature dependent. Quantitative autoradiography revealed that the density of binding sites was high in the stratum oriens and stratum radiatum of the CA1-CA4 regions of Ammon's horn. The pyramidal cell layer was faintly labelled. Binding was almost undetectable in the dentate gyrus. The highest density of sites (Bmax = 11.6 +/- 1.0 fmol/mg protein) was observed in the stratum radiatum of the CA3 region. Under the same conditions the value obtained for pituitary tissues was 20.7 +/- 2.8 fmol/mg protein. Analysis of saturation curves indicated only one class of high-affinity sites for the hippocampus (CA3; Kd = 0.28 +/- 0.03 nM) and for the pituitary (Kd = 0.29 +/- 0.08 nM). Both native GnRH and GnRH antagonist were potent competitors of binding. Fresh slices and membrane preparations from whole hippocampus confirmed these autoradiographic data and yielded affinity constants of 0.28 +/- 0.01 and 0.52 +/- 0.08 nM, respectively. In addition, a very high binding density was present in the amygdaloid complex, while binding was barely detectable in the hypothalamus. These results demonstrate that high densities of specific GnRH receptors are present in areas concerned with the regulation of behavioral functions.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of ovine pineal compounds prepared under red or green light on the activity of male rat anterior pituitaries in vitro.

A high molecular weight fraction XM100R (MW A 100,000) was prepared by ultrafiltration from ovine pineals using two different extraction methods under red light conditions (lambda greater than 600 nm). This fraction stimulates the release of radioimmunologically active luteinizing hormone (LH) of anterior pituitaries in vitro. The ultrafiltration fraction PM30R (MW greater than 30,000 and less than 100,000) was found to be radioimmunologically active only when the "Bensinger" extraction procedure was applied. However, when comparable fractions were prepared under green light and incubated with half-pituitaries, all the incubation media of the ultrafiltrated fractions, XM100R, PM30R, PM10R (MW greater than 10,000 and less than 30,000) UM2R (MW greater than 1000 and less than 10,000), UM05R (MW greater than 500 and less than 1000) and UM05F (MW greater than 500), reacted with anti-LH. This may mean that under green light conditions the high molecular weight ovine pineal compounds in XM100R are disintegrated and/or split up into small molecules which can stimulate the release of LH, or crossreact with the anti-LH serum.

Characterization of a neutral endopeptidase localized in the mitochondrial matrix of rat anterior pituitary tissue with GnRH as a substrate.

We have determined the subcellular localization of an endopeptidase activity able to degrade gonadotropin releasing hormone (GnRH) and present in the rat adenohypophysis. After fractionation of tissue homogenates in 0.25 M sucrose by differential centrifugation, about 25% of the total cellular GnRH degrading activity was found to be sedimentable and recovered from heavy (M) and light (L) mitochondrial fractions with a distribution pattern similar to that of the mitochondrial and lysosomal reference enzymes cytochrome oxidase and beta-galactosidase. Upon further fractionation on sucrose density gradients, the activity comigrated with mitochondria. The peptidase appears endowed with a structure-linked latency; the activity is low in a freshly prepared mitochondrial fraction and increases upon treatment with membrane disrupting agents in a manner similar to that of malate dehydrogenase, a component of the mitochondrial matrix. Determination of GnRH cleavage sites was performed by amino acid analysis of the fragments obtained after incubation of the peptidase with (3H)-GnRH labelled on the pyroglutamic acid residue, in presence of carboxypeptidase and peptidyldipeptidase inhibitors. The fragments were separated by ion-exchange chromatography on an Aminex Q-15S column and purified by chromatography on silica gel plates. Fragments 1-2, 1-3, 1-4, 1-5 and 1-6 were all present as early as 1 min after the beginning of incubation. Formation of each of them was inhibited to the same extent by EDTA, mersalyl acid, dithioerythritol and Na deoxycholate. The same fragmentation pattern was observed after partial purification of the enzyme by gel filtration. These data indicate that cleavage of several peptide bonds may result from a possibly single endopeptidase located in the mitochondrial matrix space.

Biphasic pattern of follicle stimulating and luteinizing hormone responses to gonadotropin-releasing hormone in vitro.

Increasing concentrations of LHRH or its active analog des-gly10 (D-ala6) LHRH induced a bimodal pattern of FSH and LH release from incubated male rat pituitaries. A low amplitude response (40% increase of LH over baseline levels) was observed for concentrations of LHRH and the agonist in the range of 10(-12) and 10(-13) M respectively. After a plateau of gonadotropin stimulation, a further high amplitude response (180-240% increase over baseline levels) occurred between 3.10(-10) and 10(-8) for LHRH and 3.10(-11) and 3.10(-9) M for des-gly10 (D-ala6) LHRH. Corresponding half maximal effective concentrations (ED50) were 2 and 0.3 nM respectively. Stimulation of FSH release closely paralleled that of LH. The low amplitude, high apparent affinity response was only obtained when the peptides were diluted in low concentrations of acidic tissue extracts. A preliminary study indicated that this method of dilution minimized peptide loss by adsorption. In addition, the low amplitude, high affinity response was never observed on pituitaries sampled from castrates. These experiments suggest the presence of two distinct populations of LHRH recognition sites on pituitary gonadotrophs. Under our experimental conditions, the appearance of the higher affinity response was dependent upon prior exposure to sex steroids. This could be due to a direct action of the steroid on the expression of a high affinity LHRH receptor.

Effects of sheep pineal fractions on the activity of male rat hypothalami in vitro.

High molecular weight substances could be isolated from sheep pineals with the "Bensinger" extraction method, followed by ultrafiltration of the waterlayer through different diaflomembranes. Two of the pineal fractions, XM100R and PM30R, stimulate the gonadotropin releasing activity of the medial basal hypothalamus (MBH). In experiments in which comparable pineal fractions were incubated without MBH and without pituitary and injected in immature mice no effect was detectable. All experiments in which a similar amount of rat cerebral cortex was used for incubation with pineal fractions did not show any activity.