Kazimierz Kochman

Binding of Cu2+, Zn2+, and Ni2+GnRH complexes with the rat pituitary receptor

Complex of copper with the gonadotropin-releasing hormone, GnRH, competed more efficiently for the GnRH receptor than native GVRH, while complexes of nickel with GnRH and zinc with GnRH had slightly lower affinity. Copper ion added to the incubation mixture inhibited the buserelin binding to the receptor.

Increased LH and FSH release from the anterior pituitary of ovariectomized rat, in vivo, by copper-, nickel-, and zinc-LHRH complexes

The effect of Cu2+, Ni2+, Zn2+ and their complexes with LHRH on the release of luteinizing hormone (LH) and follicle stimulating hormone (FSH) was estimated in in vivo experiments with the use of the method proposed by Ramirez and McCann. Ovariectomized, estradiol, and progesterone pretreated rats were injected intravenously either with LHRH alone, a metal ion alone, a mixture of metal and hormone, or a metal-LHRH complex. A metal alone or a mixture of it with LHRH did not affect gonadotropin release at all or no more than LHRH alone. However, the complex of Cu2+ with LHRH brought about a high release of LH and even higher release of FSH. This indicates that copper complex is more effective than metal-free LHRH. The nickel complex showed a similar although lesser effect. The zinc complex had similar potency to free LHRH though higher FSH-releasing ability was noticed. We conclude that copper-, nickel-, and zinc-LHRH complexes were more potent than the peptide hormone itself and promoted the FSH release in the ovariectomized, estradiol, and progesterone pretreated rats.

Modulation of luteinizing hormone subunit gene expression by intracerebroventricular microinjection of gonadotropin-releasing hormone or β-endorphin in female rats

The effects of gonadotropin-releasing hormone (GnRH), beta-endorphin and its antagonist naloxone on the expression of luteinizing hormone (LH) subunit genes and LH secretion were examined in ovariectomized and/or cycling female rats through their direct microinjection into the third cerebral ventricle, in the proximity of the hypothalamus-pituitary complex. GnRH (1 nM) induced a significant augmentation of the pituitary content of alpha mRNA when administered 15, 30 or 60 min intervals over 5 h to ovariectomized rats whereas only the 30 and 60 min intervals were effective in increasing LHbeta mRNA, and the 60 min intervals for LH release. This was in agreement with the established concept of a pulse-dependent regulation of gonadotropin synthesis and release. Hourly pulses of GnRH also increased alpha and LHbeta mRNA levels when microinjected in female cycling rats during proestrus or diestrus II. Using this model we observed a marked negative influence of hourly intracerebral microinjections of beta-endorphin on LH mRNA content and LH release in ovariectomized rats while naloxone had no effect. This suggests that endogenous beta-endorphin was unable to exert its negative action on beta-endorphin receptors that were present and responded to the ligand. The present approach would be valuable for the exploration of the mechanisms of action of beta-endorphin or other substances on the functions of the gonadotrophs.

Impaired growth hormone-releasing hormone neurons ultrastructure and peptide accumulation in the arcuate nucleus of mosaic mice with altered copper metabolism.

A progressive decrease in body weight and retarded linear growth observed in mosaic male mice with the mutation linked to X-chromosome (Atp7a(mo-ms)) raised the question whether hypophysiotropic growth axis activity may be affected in these animals. A pathologically developed median eminence ultrastructure with very low somatostatin accumulation as well as an intensive phagocytosis of growth hormone cells observed in the anterior pituitary gland raised the question whether hypothalamic growth hormone-releasing hormone (GHRH) neuronal network is also affected in mosaic mice. In this study an arcuate nucleus GHRH neurons ultrastructure as well as GHRH peptide accumulation in normal and mutant mice were compared. An electron microscopic immunocytochemical method with colloidal-gold labeling was applied to compare the ultrastructural morphology of GHRH neuron and intracellular GHRH peptide distribution. Mosaic mice exhibited a pathologically developed ultrastructure of arcuate nucleus GHRH neurons, defective intracellular peptide localization as well as reduced peptide storage. Obtained results support the crucial role of unaltered copper metabolism in physiological development of hypophysiotropic growth axis activity. Consequently, a pathologically developed GHRH hypothalamic network may impact progressive decrease in body weight and retarded length growth observed in mosaic male mice.

In vivo modulation of follicle-stimulating hormone release and β subunit gene expression by activin A and the GnRH agonist buserelin in female rats

The effects of separate and simultaneous recombinant bovine (rb) activin A and buserelin administration on the FSH release and pituitary FSH beta subunit gene expression in vivo were examined in ovariectomised, estradiol pretreated rats. The animals received a single injection of either rb activin A (50 ng), buserelin (1 micro g) or activin/buserelin (50 ng+1 micro g/0.1 ml PBS) into the jugular vein and were killed 30 min, 1, 3 and 5h later. Activin A stimulated FSH release and effect appeared 1h after injection (168% increase of controls) reaching a maximum at 3h (437% of controls). Activin A and buserelin exerted their effects with a distinct time courses: activins stimulation was not so rapid when compared with buserelin. The simultaneous administration of rb activin A and buserelin amplified FSH release (118, 309, 1006 and 779% of controls). The low dose of activin A was sufficient to elevate FSH beta mRNA level as early as 3 and 5h after administration (170 and 140%, respectively). Activin plus buserelin stimulation resulted in a higher (340 and 360% of controls) FSH beta gene expression than after their separate administration. These results suggest that activin and buserelin may act independently and synergistically in the regulation of FSH release and beta subunit mRNA level.

Growth hormone cell phagocytosis in adenohypophysis of mosaic mice: Morphological and immunocytochemical electron microscopy study

An electron microscopy immunocytochemical study was performed to determine the expression pattern of growth hormone (GH) in mosaic mutant mice adenohypophysis. In normal condition GH was restricted to the secretory granules of all growth hormone cells. Mosaic mice adenohypophysis contained growth hormone cells which have distinctive GH labeled secretory granules at the level seen in control animals. Ultrastructurally, some GH cells of mosaic mice presented abnormalities, but labeling intensity of secretory granules in these cells was always comparable to the basal condition. The striking findings presence of two forms (simple and activated) of folliculo-stellate cells (FS) in close association trough gap or tight junction with GH cells localized especially near the perivascular space. Frequently, in cytoplasm of FS cells, large clusters containing fragments of GH labeled cell were present. Additionally, the existence of large intracellular, electron-lucent spaces, with remnant cellular material in parenchyma of mosaic mutant mice adenohypophysis could suggest intensive process of GH-cell destruction. Our electron microscopy immunocytochemical results provide evidence for loss of GH cells in mosaic mice by phagocytosis. We suppose that impaired body growth observed in mosaic mutant male rats may be, at least partially, a consequence of an alteration in somatotropic axis activity. Loss of GH cells in mosaic mice by phagocytosis supported by FS cells may contribute to this effect.

Stimulation of Luteinizing Hormone Subunit Gene Expression by Pulsatile Intracerebroventricular Microinjection of Galanin in Female Rats

Although galanin, which exerts its effects both at the hypothalamic and pituitary level, has been implicated as an important neuroendocrine regulator of hypothalamic‐pituitary‐gonadal axis activity, there is a lack of data concerning its involvement in the regulation of gonadotropin subunit gene expression. To elucidate whether galanin can influence luteinizing hormone (LH) subunit mRNA content, as well as affect gonadotropin‐releasing hormone (GnRH) receptor activity, a model based on pulsatile (one pulse per hour over 5 h) galanin (1 nM) microinjections directly into the third cerebral ventricle of ovariectomized (OVX) and/or oestrogen/progesterone‐pretreated rats was used. Furthermore, to determine galanin effects on GnRH‐induced LH subunit mRNA synthesis, a cocktail of 1 nM GnRH and 1 nM galanin was coadministered in a pulsatile manner to OVX/steroid primed rats. Subsequently, to obtain data concerning the role of galanin receptors in the regulation of pituitary α (common to LH, follicle‐stimulating hormone, thyroid‐stimulating hormone) and LHβ subunit gene expression, OVX/oestrogen/progesterone rats received microinjections of 1 nM of the receptor antagonist galantide and 1 nM of galanin. In this case, both substances were administered separately, with a 30 min lag, according to which each galantide pulse always preceded a galanin pulse. Northern‐blot analysis revealed that intracerebroventricular pulsatile galanin injections were effective in stimulation of both α and LHβ subunit mRNA levels and that this effect was apparently steroid‐dependent. Moreover, galanin also up‐regulated GnRH receptor functional parameters (affinity and maximum binding capacity) but was ineffective in potentiating GnRH‐induced accumulation of both subunit mRNAs. The results from the study also indicate that galanin acts through its own receptor(s) because a receptor antagonist, galantide, significantly reduced the stimulatory effect exerted by galanin on the expression of both LH subunit genes in vivo.

Cobalt complex with GnRH stimulates the LH release and PKA signaling pathway in pig anterior pituitary cells in vitro

Metal complexes with GnRH were shown to interact with GnRH receptors in pituitary cells. In the present study we examined the effects of GnRH and its cobalt complex form (Co-GnRH) on LH secretion and generation of second messengers, namely inositol phosphates (IPs) and cAMP, in porcine pituitary cells in vitro. The cells were obtained from gilt pituitary at the pre-ovulatory phase of estrous cycle and cultured for 72 h before challenge with GnRH or Co-GnRH. Both substances induced a significant increase in LH release that was detectable after 60 min (P<0.05) of treatment, with the Co-GnRH complex being more efficient than GnRH at 180 min (P<0.01). GnRH and Co-GnRH were equally effective at 10(-8)M (P<0.01), however, at the lowest (10(-9)M) as well as the highest (10(-7)M) concentrations tested, Co-GnRH was more potent than its native counterpart (P<0.01). Interestingly, Co-GnRH revealed twice more efficient than GnRH at stimulating cAMP production, an effect which was detectable in cells after 1h-incubation (P<0.001). In contrast, while native GnRH induced a rapid increase (P<0.05) in IPs no such effect of Co-GnRH was observed. These data demonstrate that Co-GnRH and GnRH differentially effect on the signaling pathway in porcine gonadotropes and suggest that in these cells, the releasing action of Co-GnRH results from the mediation via the cAMP/protein kinase A second messenger system.

Vasoactive intestinal peptide modulates luteinizing hormone subunit gene expression in the anterior pituitary in female rat.

The direct monosynaptic pathway which exists between vasoactive intestinal peptide (VIP) and GnRH neurons in the hypothalamic preoptic area provides a neuroanatomical background for the modulatory effects of VIP exerted on GnRH neurons activity. Though central microinjection of VIP revealed its involvement in the modulation of LH release pattern, there is a lack of data concerning a possible VIP influence on the alpha and LHbeta subunit gene expression in the pituitary gland. Using a model based on intracerebroventricular pulsatile peptide(s) microinjections (1 pulse/h [10 microl/5 min] over 5 h) the effect of exogenous VIP (5 nM dose) microinjection on subunits mRNA content in ovariectomized/oestrogen-pretreated rats was studied. Subsequently, to obtain data concerning the involvement of GnRH and VIP receptor(s) in the regulation of alpha and LHbeta subunit mRNA expression, OVX/estrogen-primed rats received a pulsatile microinjections of 5 nM VIP with 3 nM antide (GnRH receptor antagonist) or 5 nM VIP with 15 nM VIP 6-28 (VIP receptor antagonist). In this case, substances were given separately with a 30 min lag according to which each antagonist pulse preceded a VIP pulse. Northern-blot analysis revealed that VIP microinjection resulted in a decreased alpha and LHbeta mRNA content in pituitary gland and this effect was dependent on GnRH receptor activity. Moreover, obtained results indicated that centrally administered VIP might operate through its own receptor(s) because a receptor antagonist, VIP 6-28, blocked the inhibitory effect of VIP exerted on both LH subunit mRNA content and LH release.

New elements in modern biological theories of aging

Generally and simply speaking, when human individuals think of how their bodies are aging, probably the most visible changes come first to their minds. One can notice more grey hair or the skin does not seem as smooth as it used to be. These are just external signs of a series of processes going on within our cells and bodily systems that together constitute normal aging. Aging (also called senescence) is an age-dependent decline in physiological function, demographically manifesting as decreased survival and fecundity with increasing age. Aging is also commonly defined as the accumulation of diverse deleterious changes occurring in cells and tissues with advancing age that are responsible for the increased risk of disease and death. The principal theories of aging are all specific of a distinct cause of aging, giving useful and deep insights for the understanding of age-related physiological changes.