Exploring Vitamin B12 Deficiency in Sleeve Gastrectomy from a Histological Study of a Cadaveric Stomach and Ileum.

INTRODUCTION: Vitamin B12 deficiency is more commonly found among patients who have undergone Roux-en-Y gastric bypass (RYGB) as compared to those with post-sleeve gastrectomies (SG). The major difference between SG and RYGB is that the latter greatly bypasses the stomach whereas the former simply reduces the gastric volume. PURPOSE: The aim of this article was to study the stomach and the distal ileum histologically in a cadaver with SG to explain the higher rate of incidences of vitamin B12 deficiency seen in patients post-RYGB relative to patients post-SG. Since the stomach is the major variable in these two procedures, we hypothesize that it has the ability to regenerate and increase its surface area to compensate for the loss of its volume in SG patients. MATERIAL AND METHODS: Tissue biopsies and hematoxylin and eosin stains were performed from various anatomical locations of the GI tract, specifically the gastric fundus, body, and antrum, and from the distal ileum of the small intestine of a cadaver with SG and another without SG (control). RESULTS: Compared with the control, the SG cadaver's gastric tissue biopsies were significant for chronic gastritis and hypertrophy of the muscularis externa layer. More importantly, parietal cell hyperplasia and deeper mucosal glands were also noted in the SG cadaver supporting the hypothesis. CONCLUSIONS: The compensatory role of an intact stomach, given its ability to regenerate parietal cells and increase its numbers in the gastric fundus and body, can be better appreciated in a gastric-sparing procedure such as SG versus RYGB in terms of limiting vitamin B12 deficiencies.

Low dosage of rimonabant leads to anxiolytic-like behavior via inhibiting expression levels and G-protein activity of kappa opioid receptors in a cannabinoid receptor independent manner.

WHAT IS KNOWN: There is an increasing number of studies demonstrating the direct effect of the cannabinoid receptor 1 (CB1) antagonist/inverse agonist rimonabant on the opioid system. The kappa opioid receptors (KORs) are well known to mediate depression- and anxiety-like behavior. Clinical studies on chronic rimonabant administration have revealed that rimonabant leads to a very similar pathophysiology, suggesting a potential impact of rimonabant on KORs. OBJECTIVES: Our objectives were to examine the putative effects of rimonabant on KOR ligand binding, G-protein activity, protein expression and how all these contribute to the development of depression- and anxiety-like behavior. RESULTS: In Chinese hamster ovary (CHO) cell membranes transfected with rat KOR (CHO-rKOR) rimonabant inhibited KOR agonist [3H]U69593 binding in the micromolar range in competition binding experiments and specifically reduced KOR basal activity at lower micromolar concentrations in [35S]GTPγS binding assays. Rimonabant significantly inhibited dynorphin (1-11)-induced [35S]GTPγS binding in micromolar range in CHO-rKOR cells, CB1 knockout (CB1 K.O.) and CB1/CB2 double knockout mouse forebrain membranes. A single dose of i.p. 0.1 mg/kg rimonabant significantly reduced dynorphin (1-11)-induced KOR G-protein activity and KOR protein expression levels 24 h following the administration in both wild type and CB1 K.O. mice forebrain. Furthermore, in elevated plus maze mice showed an anxiolytic-like effect upon rimonabant injection that could be reversed by 1 mg/kg KOR antagonist norbinaltorphimine. The anxiolytic-like effects were further confirmed with the light­dark box test. CONCLUSION: Rimonabant reduced KOR ligand binding, receptor mediated G-protein activity and protein expression level, which overall leads to altered anxiety-like behavior.

Mechanism of action of salsolinol on tyrosine hydroxylase.

Tyrosine hydroxylase (TH) is the first and rate-limiting enzyme in dopamine synthesis. Dopamine regulates TH as an end-product inhibitor through its binding to a high and low affinity site, the former being abolished by Ser40 phosphorylation only, and the latter able to bind and dissociate according to intracellular dopamine levels. Here, we have investigated TH inhibition by a dopamine metabolite found in dopaminergic brain regions, salsolinol (SAL). SAL is known to decrease dopamine in the nigrostriatal pathway and mediobasal hypothalamus, and to also decrease plasma catecholamines in rat stress models, however a target and mechanism for the effects of SAL have not been found. We found that SAL inhibits TH activity in the nanomolar range in vitro, by binding to both the high and low affinity dopamine binding sites. SAL produces the same level of inhibition as dopamine when TH is non-phosphorylated. However, it produces 3.7-fold greater inhibition of Ser40-phosphorylated TH compared to dopamine by competing more strongly with tetrahydrobiopterin, the cofactor of this enzymatic reaction. SAL's potent inhibition of phosphorylated TH would prevent TH from being fully activated to synthesise dopamine.

Thalamic neuropeptide mediating the effects of nursing on lactation and maternal motivation.

Nursing has important physiological and psychological consequences on mothers during the postpartum period. Tuberoinfundibular peptide of 39 residues (TIP39) may contribute to its effects on prolactin release and maternal motivation. Since TIP39-containing fibers and the receptor for TIP39, the parathyroid hormone 2 receptor (PTH2 receptor) are abundant in the arcuate nucleus and the medial preoptic area, we antagonized TIP39 action locally to reveal its actions. Mediobasal hypothalamic injection of a virus encoding an antagonist of the PTH2 receptor markedly decreased basal serum prolactin levels and the suckling-induced prolactin release. In contrast, injecting this virus into the preoptic area had no effect on prolactin levels, but did dampen maternal motivation, judged by reduced time in a pup-associated cage during a place preference test. In support of an effect of TIP39 on maternal motivation, we observed that TIP39 containing fibers and terminals had the same distribution within the preoptic area as neurons expressing Fos in response to suckling. Furthermore, TIP39 terminals closely apposed the plasma membrane of 82% of Fos-ir neurons. Retrograde tracer injected into the arcuate nucleus and the medial preoptic area labeled TIP39 neurons in the posterior intralaminar complex of the thalamus (PIL), indicating that these cells but not other groups of TIP39 neurons project to these hypothalamic regions. We also found that TIP39 mRNA levels in the PIL markedly increased around parturition and remained elevated throughout the lactation period, demonstrating the availability of the peptide in postpartum mothers. Furthermore, suckling, but not pup exposure without physical contact, increased Fos expression by PIL TIP39 neurons. These results indicate that suckling activates TIP39 neurons in the PIL that affect prolactin release and maternal motivation via projections to the arcuate nucleus and the preoptic area, respectively.

The CB1 receptor mediates the peripheral effects of ghrelin on AMPK activity but not on growth hormone release.

This study aimed to investigate whether the growth hormone release and metabolic effects of ghrelin on AMPK activity of peripheral tissues are mediated by cannabinoid receptor type 1 (CB1) and the central nervous system. CB1-knockout (KO) and/or wild-type mice were injected peripherally or intracerebroventricularly with ghrelin and CB1 antagonist rimonabant to study tissue AMPK activity and gene expression (transcription factors SREBP1c, transmembrane protein FAS, enzyme PEPCK, and protein HSL). Growth hormone levels were studied both in vivo and in vitro. Peripherally administered ghrelin in liver, heart, and adipose tissue AMPK activity cannot be observed in CB1-KO or CB1 antagonist-treated mice. Intracerebroventricular ghrelin treatment can influence peripheral AMPK activity. This effect is abolished in CB1-KO mice and by intracerebroventricular rimonabant treatment, suggesting that central CB1 receptors also participate in the signaling pathway that mediates the effects of ghrelin on peripheral tissues. Interestingly, in vivo or in vitro growth hormone release is intact in response to ghrelin in CB1-KO animals. Our data suggest that the metabolic effects of ghrelin on AMPK in peripheral tissues are abolished by the lack of functional CB1 receptor via direct peripheral effect and partially through the central nervous system, thus supporting the existence of a possible ghrelin-cannabinoid-CB1-AMPK pathway.

Role of salsolinol in the regulation of pituitary prolactin and peripheral dopamine release.

(R)-Salsolinol (SAL), a dopamine (DA)-related tetrahydroisoquinoline, has been found in extracts of the neuro-intermediate lobes (NIL) of pituitary glands and in the median eminence of the hypothalamus obtained from intact male rats and from ovariectomized and lactating female rats. Moreover, analysis of SAL concentrations in NIL revealed parallel increases with plasma prolactin (PRL) in lactating rats exposed to a brief (10 min) suckling stimulus after 4-h separation. SAL is sufficiently potent in vivo to account for the massive discharge of PRL that occurs after physiological stimuli (i.e. suckling). At the same time, it was without effect on the secretion of other pituitary hormones. It has been also shown that another isoquinoline derivative, 1-methyldihydroisoquinoline (1MeDIQ), which is a structural analogue of SAL, can dose-dependently inhibit the in-vivo PRL-releasing effect of SAL. Moreover, 1MeDIQ can inhibit the elevation of plasma PRL induced by physiological stimuli, for example suckling, or in different stressful situations also. 1MeDIQ also has a psycho-stimulant action, which is fairly similar to the effect of amphetamine, i.e. it induces an increase in plasma catecholamine concentrations. It is clear from these data that this newly discovered endogenous compound could be involved in regulation of pituitary PRL secretion. It has also been observed that SAL is present in peripheral, sympathetically innervated organs, for example the atrium, spleen, liver, ovaries, vas deferens, and salivary gland. Furthermore, SAL treatment of rats results in dose-dependent and time-dependent depletion of the DA content of the organs listed above without having any effect on the concentration of norepinephrine. More importantly, this effect of SAL can be completely prevented by amphetamine and by 1MeDIQ pretreatment. It is clear there is a mutual interaction between SAL, 1MeDIQ, and amphetamine or alcohol, not only on PRL release; their interaction with catecholamine "synthesis/metabolism" of sympathetic nerve terminals is also obvious.

Effect of concomitant progesterone administration or the effect of removal of estrogen capsule on changes caused by long-term estrogen treatment in pituitary VIP immunoreactivities.

In the anterior pituitary besides the classical tropic hormones, peptides of a small molecular weight are also synthesized. One of them is the vasoactive intestinal polypeptide (VIP). VIP immunoreactivity is readily detected in human and monkey pituitaries; however, in the rat VIP immunoreactive cells were observed in about 50% of intact rats. In estrogen treated rats VIP immunoreactive cells were observed in the anterior pituitary of all animals. In this work, we have examined the effect of long-term sexual steroid treatments on the VIP immunoreactivity of the anterior pituitary using diethylstilbestrol (DES) or progesterone (P) filled capsules. The effectiveness of steroid treatments was tested by the measurement of plasma prolactin (PRL) level and by the appearance of prolactinoma. DES enhanced the plasma PRL level and 5 months later it induced prolactinomas, the concomitant P treatment prevented both the elevation of plasma PRL level and the formation of prolactinomas. These results indicated that there was enough steroid in the capsules. There was a positive correlation between the duration of DES influence and the number of VIP immunoreactive cells. Two months after the implantation of DES there was a considerable number of VIP cells in the anterior pituitary, and 5 months after implantation the number of VIP cells was greatly increased so as to form a VIP-oma. Concomitant implantation of P prevented the formation of VIP-oma. Two months after the implantation, the DES capsule was removed. Already 2 months after removal the number of VIP cells approximated to the control level. It has been concluded that P can prevent the undesired effect of DES not only on the PRL, but on the VIP immunoreactivity as well.

Tuberoinfundibular peptide of 39 residues is activated during lactation and participates in the suckling-induced prolactin release in rat.

Tuberoinfundibular peptide of 39 residues (TIP39) and the PTH-2 receptor (PTH2R) constitute a peptide-receptor neuromodulator system. Based on the abundance of TIP39 fibers and axonal terminals as well as PTH2R-containing neurons and their processes in the hypothalamic para- and periventricular and arcuate nuclei TIP39 has been suggested to play a role in neuroendocrine regulation. We showed previously that TIP39 expression decreased dramatically by adulthood. In the present study, using in situ hybridization histochemistry, real-time RT-PCR, and immunohistochemistry, we found that TIP39 mRNA and peptide expression levels are markedly elevated in the posterior intralaminar complex of the thalamus (PIL) of lactating dams, one of the three locations of TIP39-containing cell bodies in the brain. In addition, in mother rats, these TIP39 neurons showed Fos expression in response to pup exposure. Transection of TIP39 fibers originating in the PIL resulted in an ipsilateral disappearance of TIP39 immunoreactivity throughout the mediobasal hypothalamus of mother rats, suggesting that TIP39 fibers there arise from the PIL. To elucidate the function of TIP39 activation in dams, mothers separated from their pups for 4 h on postpartum d 9 received injection of a PTH2R antagonist into the lateral ventricle 5 min before returning the pups. Blood samples were taken seven times during the experimental period through jugular cannulae. The PTH2R antagonist administered in two different concentrations markedly inhibited suckling-induced elevation of plasma prolactin levels in a dose-dependent manner. These results suggest that TIP39 neurons in the PIL may regulate suckling-induced prolactin release in rat dams.

Nesfatin-1/NUCB2 may participate in the activation of the hypothalamic-pituitary-adrenal axis in rats.

Nesfatin-1 is an anorexigenic peptide originating from nucleobinding-2 (NUCB2) protein. Nesfatin-1/NUCB2-immunoreactive neurons are present in the hypothalamic paraventricular nucleus, the center of the stress-axis, and in the medullary A1 and A2 catecholamine cell groups. The A1 and A2 cell groups mediate viscerosensory stress information toward the hypothalamic paraventricular nucleus. They contain noradrenaline, but subsets of these neurons also express prolactin-releasing peptide acting synergistically with noradrenaline in the activation of the hypothalamic paraventricular nucleus during stress. We investigated the possible role of nesfatin-1/NUCB2 in the stress response. Intracerebro-ventricular administration of nesfatin-1 elevated both plasma adrenocorticotropin and corticosterone levels, while in vitro stimulation of the hypophysis was ineffective. Single, long-duration restraint stress activated (Fos positivity) many of the nesfatin-1/NUCB2-immunoreactive neurons in the parvocellular part of the hypothalamic paraventricular nucleus, evoked nesfatin-1/NUCB2 mRNA expression in the parvocellular part of the paraventricular nucleus and in the A1, but not in the A2 cell group. Nesfatin-1/NUCB2 was shown to co-localize in a high percentage of prolactin-releasing peptide producing neurons, in both medullary catecholamine cell groups further supporting its involvement in the stress response. Finally, bilateral adrenalectomy evoked an increasing nesfatin-1/NUCB2 mRNA expression, indicating that it is under the negative feedback of adrenal steroids. These data provide the first evidence for possible participation of nesfatin-1/NUCB2 in the stress-axis regulation, both at the level of the brainstem and in the hypothalamus.

Dephosphorylation/inactivation of tyrosine hydroxylase at the median eminence of the hypothalamus is required for suckling-induced prolactin and adrenocorticotrop hormone responses.

We have recently found that dopamine (DA) released from terminals of the hypothalamic neuroendocrine dopamine (NEDA) neurons plays a role not only in prolactin (PRL), but also in adrenocorticotrop hormone (ACTH) secretion, without having any influence on alpha-melanocyte-stimulating hormone (alpha-MSH) release in lactating dams. The aim of our present studies was to further investigate this DAerg regulation of ACTH using consecutively applied physiological stimulation (suckling) and pharmacological inhibition of the rate-limiting enzyme of DA synthesis (tyrosine hydroxylase, TH) by alpha-methyl-p-tyrosine (alpha-MpT) that acutely affect secretion of these pituitary hormones during lactation. Following 4h separation period, two experimental groups were formed. In the first group, lactating rats were assembled with their litters for 60 min prior to alpha-MpT. In the second group, the alpha-MpT was injected first and 60 min later suckling stimulus was applied. Plasma samples were taken in every 15 min during the 90 min experimental period. Concentrations of plasma PRL, ACTH and alpha-MSH were measured by specific RIAs. Both stimuli applied in the first sequence, significantly elevated plasma PRL and ACTH levels in separated lactating dams, without having any effect on alpha-MSH secretion. Suckling applied in the first sequence was able to block the alpha-MpT-induced elevation of ACTH secretion, while PRL response was also significantly attenuated. alpha-MpT pretreatment prevented both PRL and ACTH responses to suckling stimulus. Investigating the dephosphorylation/inactivation of TH in the arcuate nucleus-ME (TIDA) regions, no pTH-immunoreactive perikarya or terminals can be found in continuously suckled dams. In contrast, after 4h separation of the mothers from their litters, pTH-immunoreactivity can be clearly visualized in the external zone of ME. In alpha-MpT pretreated mothers following 4h separation no pTH positive terminals are visible. No changes in the TH immunostaining can be observed in any of these experimental groups. In conclusion, dephosphorylation/inactivation of TH (the rate-limiting enzyme of the DA biosynthesis) in NEDA neurons is required for suckling-induced PRL and ACTH responses.

Plasma prolactin concentrations and copulatory behavior after salsolinol injection in male rats.

PURPOSE: The dopamine-derived endogenous compound, R-salsolinol (SAL), was recently identified as a putative endogenous prolactin (PRL)-releasing factor. However, how SAL influences copulatory behavior is unknown. In this study, we examined the relationship between SAL and copulatory behavior in male rats. METHODS: Male Sprague-Dawley rats administered SAL were exposed to female rats in estrus, the plasma PRL concentration was measured, and the behavioral frequency and time during copulatory behavior were noted. RESULTS: In the control and SAL groups, plasma PRL concentrations at 15 min before exposure to the female were 7.3 ± 2.0 and 8.0 ± 1.5 ng/ml, respectively. Moreover, plasma PRL concentrations in males immediately after exposure to the female were 7.4 ± 1.2 and 68.0 ± 5.9 ng/ml, respectively (P < 0.05). All (8/8) of the control animals ejaculated in the presence of the female, whereas only 33% (2/6) of the SAL group ejaculated. An increasing tendency for mount latency and intromission latency and a decreasing tendency for intromission frequency were observed in the SAL group. CONCLUSIONS: Copulatory behavior was inhibited in male rats after SAL injection, suggesting that SAL is a copulatory behavior inhibiting factor.

Dopamine-regulated adrenocorticotropic hormone secretion in lactating rats: functional plasticity of melanotropes.

Pro-opiomelanocortin (POMC) is processed to adrenocorticotropic hormone (ACTH) and beta-lipotropin in corticotropes of the anterior lobe, and to alpha-melanocyte-stimulating hormone (alpha-MSH) and beta-endorphin in melanotropes of the intermediate lobe (IL) of the pituitary gland. While ACTH secretion is predominantly under the stimulatory influence of the hypothalamic factors, hormone secretion of the IL is tonically inhibited by neuroendocrine dopamine (NEDA) neurons. Lobe-specific POMC processing is not absolute. For example, D(2) type DA receptor (D2R)-deficient mice have elevated plasma ACTH levels, although it is known that corticotropes do not express D2R(s). Moreover, observations that suckling does not influence alpha-MSH release, while it induces an increase in plasma ACTH is unexplained. The aim of the present study was to investigate the involvement of the NEDA system in the regulation of ACTH secretion and the participation of the IL in ACTH production in lactating rats. Untreated and estradiol (E(2))-substituted ovariectomized (OVX) females were also studied. The concentration of ACTH in the IL was higher in lactating rats than in OVX rats, while the opposite change in alpha-MSH level of the IL was observed. DA levels in the IL and the neural lobe were lower in lactating rats than in OVX rats. Suckling-induced ACTH response was eliminated by pretreatment with the DA receptor agonist, bromocriptine (BRC). Inhibition of DA biosynthesis by alpha-methyl-p-tyrosine (alphaMpT) and blockade of D2R by domperidone (DOM) elevated plasma ACTH levels, but did not influence plasma alpha-MSH levels in lactating rats. The same drugs had opposite effects in OVX and OVX + E(2) animals. In lactating mothers, BRC was able to block ACTH responses induced by both alphaMpT and DOM. Surgical denervation of the IL elevated basal plasma levels of ACTH. Taken together, these data indicate that melanotropes synthesize ACTH during lactation and its release from these cells is regulated by NEDA neurons.

Time-dependent changes in cardiovascular function during copulation in male rats.

PURPOSE: Sudden cardiac death after ejaculation has been reported in humans and highlights the important relationship between sexual behavior and the heart. The rat is an extremely useful animal model for investigating reproductive function in male mammals. In this study, we examined the relationship between autonomic nervous system activity and the circulatory system during sexual behavior in male rats. METHODS: Male Wistar-Imamichi rats were exposed to female rats in estrous and heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), and plasma noradrenaline (NA) and adrenaline (Ad) concentrations were measured by telemetry during copulation. RESULTS: The resting HR was 365.5 ± 18.4 beats/min (mean ± SE), which increased to 531.2 ± 21.1 beats/min at ejaculation and decreased to 404.6 ± 30.7 beats/min 1 min after ejaculation. At rest, SBP and DBP were 123.8 ± 6.6 and 81.5 ± 4.1 mmHg, respectively, which increased to 154.5 ± 5.9 and 112.1 ± 7.3 mmHg at ejaculation. Baseline plasma Ad and NA concentrations were 151.6 ± 32.0 and 248.5 ± 22.5 pg/ml, respectively, and these increased to 393.8 ± 89.9 and 792.7 ± 154.0 pg/ml at ejaculation, after which they decreased to resting levels. The rate of increase in NA at ejaculation differed significantly from that of Ad. CONCLUSIONS: The load on the circulatory and autonomic nervous systems is controlled by a rapid decrease in HR and NA concentration immediately after ejaculation, such that the male rat is prepared for the next copulation.

A simple reaction kinetic model of rapid (G protein dependent) and slow (beta-Arrestin dependent) transmission.

In this paper the qualitative dynamic behavior of reaction kinetic models of G protein signaling is examined. A simplified basic G protein signaling structure is defined, which is extended to be able to take the effect of slow transmission, RGS mediated feedback regulation and ERK-phosphatase mediated feedback regulation into account. The resulting model gives rise to an acceptable qualitative approximation of the G protein dependent and independent ERK activation dynamics that is in good agreement with the experimentally observed behavior.

Demonstration of autoantibody binding to muscarinic acetylcholine receptors in the salivary gland in primary Sjögren's syndrome.

A significant pathogenetic role of antimuscarinic acetylcholine receptor-3 (anti-m3AChR) autoantibodies in primary Sjögren's syndrome (pSS) has been suggested. However, the binding of these antibodies to the receptors in the target tissues has not yet been demonstrated. In this study, the binding characteristics of pSS sera and anti-m3AChR-monospecific sera affinity-purified from pSS patients to labial salivary gland samples from healthy subjects were studied with light- and electron microscopy. Furthermore, the ultrastructural localisation of in vivo deposited antibodies in pSS salivary glands was also investigated. Light microscopic immunohistochemistry revealed the binding of the anti-m3AChR-specific sera to the membrane of acinar cells. Similar reaction end-products were observed in the pSS salivary gland epithelial cell membranes. With electron microscopy, the autoantibody binding was observed to be localised to the junctions of epithelial cell membranes with nerve endings, both in normal and pSS glands. The results indicate that anti-m3AChR antibodies bind to the receptors in the salivary glands.

The response of plasma catecholamines in rats simultaneously exposed to immobilization and painful stimuli.

Immobilization represents a strong stressor inducing a profound increase in plasma epinephrine and norepinephrine levels. We have previously demonstrated that a subcutaneous injection of formalin (0.2 mL of 4% solution/100 g bw) attenuated the immobilization-induced elevation of plasma epinephrine levels in rats. In the present study, we investigated whether other painful and stressful stimuli, such as capsaicin, hydrochloric acid, mechanical pressure, heat, and cold, might also attenuate the increase of plasma epinephrine in rats exposed to acute immobilization stress. With the exception of formalin, all of the painful stimuli applied failed to attenuate the increase of plasma epinephrine levels in immobilized animals. Our data suggest that the attenuation of an immobilization-induced increase in plasma epinephrine levels is specific for subcutaneous formalin administration.

Prolactin response to formalin is related to the acute nociceptive response and it is attenuated by combined application of different stressors.

Subcutaneous injection of diluted formalin (0.2 ml of 4% solution/100 g BW) can influence the increase of plasma epinephrine levels in rats exposed to exteroceptive (handling, immobilization), as well as to interoceptive stressors (insulin-induced hypoglycemia), without having any effect on norepinephrine release. In the present studies, the effect of the above-mentioned stressors has been investigated on formalin-induced prolactin (PRL) and corticosterone secretion. Administrations of formalin via chronically implanted subcutaneous cannula into the hind limb without handling induce an immediate increase in both plasma PRL and corticosterone levels. While PRL concentration reaches its peak value within 5 min then returns to the basal level by the end of the 30th min, corticosterone level also starts to rise immediately after formalin administration reaching its highest concentration within 15-30 min, but it remains at this high level during the next 60 min, then it declines and returns to the pre-injection level. Application of formalin to animals exposed to different heterotypic stressors (like handling or insulin-induced hypoglycemia) produces an attenuated PRL response, while plasma corticosterone levels induced by the same nociceptive component remained unchanged. Combinations of formalin injection with immobilization also show an attenuated PRL response. The present data indicate that plasma PRL response to formalin is related to its acute nociceptive phase, and application of different stressors prior to formalin injection significantly attenuate plasma PRL levels, while it does not influence corticosterone responses.

Salsolinol and the peripheral sympathetic activity: the effects of hypophysectomy, adrenalectomy and adrenal medullectomy.

The endogenous isoquinoline salsolinol (SALS) is a recently identified prolactin (PRL) releasing factor, a selective and potent stimulator of PRL secretion both in vivo and in vitro. SALS decreased the peripheral tissue dopamine (DA) level dose dependently, consequently increased the NE/DA ratio, indicating reduced release of newly formed norepinephrine (NE) from sympathetic terminals. The aim of our study was to investigate the effect of adrenal medullectomy (MEDX), adrenalectomy (ADX) and hypophysectomy (HYPOX) on the action of SALS on the PRL secretion, and on the catecholamine concentration of the selected sympathetically innervated peripheral tissues (atrium, spleen, etc). The experiments were done in male rats of 200-300 g body weight kept in air conditioned room with regular lighting. We used high-pressure liquid chromatography with electrochemical detection (HPLC-EC) for measurement of NE and DA concentrations, and radioimmunoassay for prolactin measurement. In MEDX as well as in ADX rats, SALS (25 mg/kg i.p.) was able to reduce DA level and increase the NE/DA ratio. The changes of prolactin secretion (increase by SALS) were not affected either by ADX or MEDX. Therefore the presence of the adrenal gland is not required for the changes of prolactin secretion, nor for the reduction of peripheral sympathetic activity induced by SALS. Investigating the possible effect of pituitary hormones on the peripheral sympathetic system, the action of SALS has been tested in HYPOX rats. We have found that the effect of SALS on peripheral sympathetic terminals is not affected by HYPOX, consequently the role of pituitary hormones in the effect of SALS on the peripheral catecholamine metabolism may be excluded.

Effect of local (intracerebral and intracerebroventricular) administration of tyrosine hydroxylase inhibitor on the neuroendocrine dopaminergic neurons and prolactin release.

BACKGROUND AND PURPOSE: Hypothalamic dopamine (DA), the physiological regulator of pituitary prolactin (PRL) secretion, is synthesized in the neuroendocrine dopaminergic neurons that projects to the median eminence and the neurointermediate lobe of the pituitary gland. The rate-limiting step of DA biosynthesis is catalyzed by the phosphorylated, therefore activated, tyrosine hydroxylase (TH) that produces L-3,4-dihydroxy-phenylalanine from tyrosine. The aims of our present study were to investigate 1. the effect of local inhibition of the DA biosynthesis in the hypothalamic arcuate nucleus on PRL release, and to get 2. some information whether the phosphorylated TH is the target of enzyme inhibition or not. METHODS: A TH inhibitor, alpha-methyl-p-tyrosine was injected either intracerebro-ventricularly or into the arcuate nucleus of freely moving rats and plasma PRL concentration was measured. Immunohistochemistry, using antibodies raised against to native as well as phosphorylated TH were used to compare their distributions in the arcuate nucleus-median eminence region. RESULTS: Intracerebro-ventricular administration of alpha-methyl-p-tyrosine has no effect, unlike the intra-arcuatus injection of enzyme inhibitor resulted in a slight but significant elevation in plasma PRL. Parallel with this, the level of DA and DOPAC were reduced in the neurointermediate lobe while no change in norepinephrine concentration can be detected indicating a reduced biosynthesis of dopamine following TH inhibition. On the other hand, systematic application of the alpha-methyl-p-tyrosine that inhibits TH activity located in DA terminals of the median eminence and the neurointermediate lobe, resulted in the most significant elevation of PRL. CONCLUSION: Our results suggest that alpha-methyl-p-tyrosine administered close to the neuroendocrine dopaminergic neurons was able to inhibit only a small proportion of the TH. Moreover, it also indicate that the majority of the activated TH can be found in the axon terminals of dopaminergic neurons, therefore, the DA released into the pituitary portal circulation is synthesized at this site.

Somatostatin analogues stimulate p27 expression and inhibit the MAP kinase pathway in pituitary tumours.

OBJECTIVES: Somatostatin (SST) analogues play an important role in the medical management of somatotroph pituitary adenomas and new agonists have the potential to be effective in a wider group of pituitary and other tumours. The anti-proliferative effect of SST occurs through multiple mechanisms, one of which is cell-cycle arrest, where p27, a cyclin-dependent kinase inhibitor, is an important regulator. We hypothesised that SST may upregulate p27 protein levels and downregulate the MAP kinase pathway in these tumours. METHODS: Human pituitary adenoma cells and rat pituitary cell line (GH3) were cultured and treated in vitro with octreotide and the broad-spectrum SST agonist SOM230 (pasireotide). Immunoblotting for p27 and phospho-ERK (pERK) was performed and proliferation assessed by [3H]-thymidine incorporation. Histological samples from acromegalic patients treated with octreotide before surgery were immunostained for p27 and compared to samples from untreated patients matched for sex, age, tumour size, extension and invasiveness. RESULTS: We detected upregulation of p27 protein levels with SST analogue treatment in vitro in human pituitary adenoma samples. pERK1/2 was inhibited by SST analogues in both the human samples and GH3 cells. SST and its analogues inhibited the proliferation of GH3 cells. p27 immunostaining was stronger in samples from patients with longer preoperative octreotide treatment (more than 6 months) than in samples from patients with shorter treatment periods. CONCLUSIONS: This study demonstrates that SST-mediated growth inhibition is associated with the downregulation of pERK and upregulation of p27. More potent and broader-spectrum SST analogues are likely to play an increasing role in the treatment of tumours, where the MAP kinase pathway is overactivated.