Histamine is involved in the regulation of collagen content in cultured heart myofibroblasts via H2, H3 and H4 histamine receptors.

Histamine is involved in the regulation of collagen metabolism during healing following a myocardial infarction; however, its effects on the intact heart tissue is unknown. The aim of the present study was to determine whether histamine may influence collagen content in cells isolated from intact heart, and to identify the histamine receptor involved in the regulation of collagen deposition. Cells were isolated from intact rat hearts and subjected to identification by flow cytometry. The effects of histamine and its receptor agonists and antagonists were investigated. The heart cells were found to be actin, desmin and vimentin positive. Histamine (used at a concentrations of 1x10-10-1x10-5 M) increased collagen content within the culture and increased the expression of α1 chain of the procollagen type III gene. The H2, H3 and H4 receptor inhibitors ranitidine, ciproxifan and JNJ 7777120 blocked the effect of histamine on collagen content. All tested histamine receptor agonists, viz. 2-pyridylethylamine dihydrochloride (H1 receptor agonist), amthamine dihydrobromide (H2 receptor agonist), imetit (H3 receptor agonist) and 4-methylhistamine hydrochloride (H4 receptor agonist), elevated collagen content within the heart myofibroblast cultures. The cells isolated from the intact heart were identified as myofibroblasts. Thus, the results of the present study showed that histamine augmented collagen content in the heart myofibroblast culture by activation of three histamine receptors (H2, H3 and H4). The effect of the amine was also dependent on the activation of collagen type III gene expression.

Anti-inflammatory and pro-healing impacts of exendin-4 treatment in Zucker diabetic rats: Effects on skin wound fibroblasts.

Using male Zucker diabetic fatty (ZDF) rats implanted subcutaneously with polyethylene mesh pieces stimulating granulation tissue development, we investigated the effects of the in vivo and in vitro treatment with exendin-4, a glucagon-like peptide-1 agonist displaying a variety of antidiabetic actions, on the markers of metabolism, inflammation, and healing in addition to skin wound fibroblast/myofibroblast activities. Exendin-4 at increasing doses of 3-10 µg/kg or 0.9% saline was injected daily to ZDF rats pre-implanted with the mesh for 3 weeks. Then, fibroblasts/myofibroblasts isolated from the granulation tissue in both groups were further exposed in vitro to exendin-4 at concentrations of 0-100 nmol/l. After a 3-week administration period, cumulative food and water intake and body weight were reduced significantly. The serum and fibroblast culture medium C-reactive protein (CRP) concentrations and matrix metalloprotease-9/tissue matrix metalloproteinase inhibitor-1 (MMP-9/TIMP-1) ratio in the fibroblast culture medium were diminished significantly in the exendin-4 pretreated group, indicating the increased expression of anti-inflammatory and pro-healing biomarkers. In vivo exendin-4 treatment also increased the number of living fibroblasts/myofibroblasts in cell cultures. The subsequent in vitro exposure to exendin-4 significantly increased metabolic activity and total collagen content in fibroblast/myofibroblast colonies derived from exendin-4-pretreated rats but reduced the number of viable cells. A cytotoxic effect was noted at the highest exendin-4 concentrations used. To conclude, the treatment of diabetic rats with exendin-4 had beneficial effects on systemic and tissue metabolic, inflammatory, and healing markers and on fibroblast functions crucial for wound repair but showed some cytotoxicity on these cells.

The role of the cAMP/PKA signalling pathway in the inhibitory influence of melatonin on oxytocin and vasopressin secretion from the rat hypothalamo-neurohypophysial system.

INTRODUCTION: Melatonin was found to inhibit forskolin-stimulated oxytocin (OT) and vasopressin (VP) release in vitro. The purpose of the present investigation was to evaluate the contribution of the cyclic 3',5'-adenosine monophosphate/protein kinase A (cAMP/PKA) signalling pathway in melatonin-dependent inhibition of OT and VP secretion from the rat hypothalamo-neurohypophysial (H-NH) system in vitro. MATERIAL AND METHODS: The H-NH explants were placed in 1 ml of normal Krebs-Ringer (nK-R) buffer and first preincubated for 30 min in control buffer or in the presence of PKA inhibitor, i.e. cAMPS-Rp or H-89. Next, they were incubated in nK-R buffer {fluid F1} and then in buffer as F1 enriched with melatonin (10-9 M or 10-7 M) and/or PKA activator, i.e. cAMP analogue (8-Br-cAMP), or their vehicles {fluid F2}. After 20 min of incubation in fluid F1 and then F2, the media were collected and frozen, to be assayed for OT and VP by the RIA. RESULTS: 8-Br-cAMP increased OT and VP secretion when the H-NH explants were preincubated in control medium, while PKA inhibitors eliminated its stimulatory effect on OT and VP release. Melatonin (10-7 M) diminished basal OT and VP output from the H-NH system, and inhibited (at both concentrations studied) the cAMP analogue-stimulated release of both neurohormones under control conditions. The effect of melatonin on OT and VP release was completely blocked when cAMPS-Rp, but not H-89, was used to disrupt the cAMP/ /PKA pathway. CONCLUSIONS: Melatonin employs the cAMP/PKA signalling pathway to inhibit OT and VP secretion from the rat H-NH system; nonethe-less, other cAMP-mediated mechanisms are not excluded.

The role of histamine in the regulation of the viability, proliferation and transforming growth factor ß1 secretion of rat wound fibroblasts.

BACKGROUND: Inflammation mediators play a regulatory role in repair processes. The study will examine the influence of histamine on wound fibroblast metabolic activity, viability, proliferation, and TGFß1 secretion. The study also will identify the histamine receptor involved in regulation of the tested repair processes. METHODS: Fibroblasts were obtained from the granulation tissue of wounds or intact dermis of rats. The MTT and BrdU assays were used to examine the effect of histamine (10-8M-10-4M) on the viability and metabolic activity of fibroblasts, and on their proliferative capacity. The influence of histamine receptor antagonists (i.e., ketotifen, ranitidine, ciproxifan and JNJ7777120) and agonists (2-pyridylethlamine dihydrochloride, amthamine dihydrobromide) was also investigated. The TGFß1 and histamine receptors H1 were evaluated by enzyme-linked immunosorbent assay. RESULTS: Histamine significantly increased granulation tissue fibroblast viability and metabolic activity at 10-8 and 10-6M but did not change their proliferative activity. Only the blockade of the H1 receptor removed this effect of histamine. H1 receptor agonist (2-pyridylethlamine dihydrochloride) increased cell viability, thereby mimicking histamine action. Both Histamine (10-4M) and 2-pyridylethlamine dihydrochloride increased TGFß1 concentration in cell culture medium. However, ketotifen blocked histamine-induced augmentation of TGFß1. H1 receptor expression on wound fibroblasts was confirmed. CONCLUSION: The regulatory influence of histamine on wound fibroblast function (viability/metabolic activity or secretion of TGFß1) is dependent on H1 receptor stimulation. Contrary to wound fibroblasts, these cells express a very low level of H1 receptors when isolated from intact dermis and histamine is unable to modify their metabolic activity.

The role of melatonin membrane receptors in melatonin-dependent oxytocin secretion from the rat hypothalamo-neurohypophysial system - an in vitro and in vivo approach.

INTRODUCTION: Melatonin exerts its biological role acting mainly via G protein-coupled membrane MT1 and MT2 receptors. To determine whether a response of oxytocinergic neurons to different concentrations of melatonin is mediated through membrane MT1 and/or MT2 receptors, the effect of melatonin receptors antagonists, i.e. luzindole (a non-selective antagonist of both MT1 and MT2 receptors) and 4-phenyl-2-propionamidotetralin (4-P-PDOT - a selective antagonist of MT2 receptor), on melatonin-dependent oxytocin (OT) secretion from the rat hypothalamo-neurohypophysial (H-N) system, has been studied both in vitro and in vivo. MATERIAL AND METHODS: For in vitro experiment, male rats served as donors of the H-N explants, which were placed in 1 ml of normal Krebs-Ringer fluid (nKRF) heated to 37oC. The H-N explants were incubated successively in nKRF {fluid B1} and incubation fluid as B1 enriched with appropriate concentration of melatonin, i.e. 10-9 M, 10-7 M, or 10-3 M and luzindole or 4-P-PDOT, or their vehicles (0.1% ethanol or DMSO) {fluid B2}. After 20 minutes of incubation in fluid B1 and then B2, the media were collected and immediately frozen before OT estimation by the RIA. The OT secretion was determined by using the B2/B1 ratio for each H-N explant. During in vivo experiment, rats were given an intracerebroventricular (i.c.v.) infusion of 5 mL luzindole or 4-P-PDOT, or their solvent (0.1% DMSO) and 10 minutes later the next i.c.v. infusion of 5 mL of either melatonin solution (10-7 M) or its vehicle (0.1 % ethanol in 0.9% sodium chloride). RESULTS: Melatonin at a concentration of 10-3 M significantly stimulated, while at a concentration of 10-9 M had no effect on, oxytocin secretion from the rat H-N system in vitro, also when luzindole or 4-P-PDOT was present in a medium. On the other hand, melatonin at a concentration of 10-7 M diminished this neurohormone output from an isolated H-N system and into the blood. Luzindole significantly suppressed such melatonin action, while 4-P-PDOT did not change the inhibitory influence of 10-7 M melatonin on oxytocin release, both in vitro and in vivo. CONCLUSIONS: The present study demonstrates that an inhibitory effect of 10-7 M melatonin on oxytocin secretion from the rat H-N system is mediated through a subtype MT1 membrane receptor and its action is independent of subtype MT2 receptor. However, for the stimulatory effect of pharmacological concentration (10-3 M) of the pineal hormone on oxytocin release, probably mechanisms other than membrane MT1/MT2 receptor(s)-dependent are involved. (Endokrynol Pol 2016; 67 (5): 507-514).

Forskolin-stimulated vasopressin and oxytocin release from the rat hypothalamo-neurohypophysial system in vitro is inhibited by melatonin.

INTRODUCTION: Previous in vivo and in vitro studies have shown that melatonin changes vasopressin (AVP) and oxytocin (OT) secretion from the rat neurohypophysis. Additionally, melatonin is known to inhibit the forskolin-induced (forskolin is a strong adenylyl cyclase - AC activator) increase in cAMP accumulation in the rat pituitary. To determine whether the possible response of vasopressinergic and/or oxytocinergic neurones to melatonin could be mediated through a cAMP-dependent mechanism, the effect of different concentrations of melatonin (i.e. 10-11, 10-9, 10-7, 10-5 and 10-3 M) on forskolin-stimulated AVP and OT release from the rat hypothalamo-neurohypophysial (H-NH) system was studied in vitro. MATERIAL AND METHODS: Male rats served as donors of the H-NH explants, which were placed in 1 mLof normal Krebs-Ringer fluid (nKRF), heated to 37oC and constantly gassed with carbogen (95% O2 and 5% CO2). The H-NH explants were incubated successively in nKRF {fluid B1} and incubation fluid as B1 enriched with an appropriate concentration of melatonin, i.e. 10-11 - 10-3 M and/or forskolin (at a concentration of 10-5 M) or their vehicles (0.1% ethanol or DMSO) {fluid B2}. After 20 min incubation in fluid B1 and next B2, the media were collected and immediately frozen before AVP and OT estimation by the RIA. The AVP and OT secretion was determined by using B2/B1 ratio for each H-NH explant. RESULTS: We have demonstrated that the highly effective AC activator - forskolin significantly stimulated both AVP and OT release from isolated rat H-NH system. Such an effect of forskolin was reduced by melatonin at concentrations of 10-9, 10-7 and 10-5 M. The strongest effect was exerted by this hormone at a concentration of 10-7 M, which inhibited not only forskolin-stimulated, but also basal, AVP and OT release. On the contrary, the highest studied concentration (i.e. 10-3 M) of melatonin stimulated both AVP and OT basal release, but when forskolin was present in the medium melatonin at such a concentration remained inactive in modifying these hormones release from the H-NH system in vitro. CONCLUSIONS: Our present results demonstrate that in the male rat: 1. The influence of melatonin on the vasopressinergic and oxytocinergic neurones activity is mediated partly through a cAMP-dependent mechanism. 2. The effect of melatonin in this respect depends on its concentration.

Oxytocin and vasopressin secretion from the rat hypothalamo-neurohypophysial system is stimulated by triptorelin.

INTRODUCTION: Several observations have suggested that the secretion of neurohypophysial hormones could be modified by gonadotropin- releasing hormone (GnRH). Since, in medical practice, more often than GnRH itself, its analogues are used, the present study was undertaken to investigate the influence of the GnRH agonist - triptorelin on oxytocin (OT) and vasopressin (AVP) release from the rat hypothalamo-neurohypophysial (H-N) system both in vitro and in vivo. MATERIALS AND METHODS: Male rats served as donors of the H-N explants, which were placed in 1 mL of Krebs-Ringer fluid (nKRF) and incubated successively in: 1 - nKRF (B1); 2 - incubation fluid as B1 enriched with an excess amount (56 mM) of K(+) (S1); 3 - incubation fluid as B1 enriched with an appropriate concentration of triptorelin, i.e., 10(-11) - 10(-5) M (B2); and 4 - incubation fluid as S1 enriched with the same concentrations of triptorelin (S2). After 20 minutes of incubation, each medium (B1, S1, B2, S2) was collected and frozen before OT and AVP estimation by the RIA. During in vivo experiment, animals were infused intracerebroventricularly (icv) with triptorelin, at a concentration of 10(-7) M, and 20 minutes later they were decapitated. The neurohypophysis was dissected from the brain and blood plasma samples were collected and frozen for further OT and AVP RIA assays. RESULTS: The GnRH agonist - triptorelin stimulates both OT and AVP release from isolated H-N system at concentrations of 10(-9)-10(-5) M. The strongest effect was displayed by triptorelin at a concentration of 10(-7) M. Under the conditions of K(+) stimulation, triptorelin affects neither OT, nor AVP secretion in vitro. When infused icv, triptorelin, at a concentration of 10(-7) M, significantly stimulated both OT and AVP secretion into the blood. CONCLUSIONS: Triptorelin may play a role as a neuromodulator contributing to the functional regulation of OT and AVP secretion in the rat.

Hypothalamic gonadotropin-releasing hormone receptor activation stimulates oxytocin release from the rat hypothalamo-neurohypophysial system while melatonin inhibits this process.

The present study was undertaken to investigate the influence of gonadotropin-releasing hormone (GnRH) and its agonist and antagonist on oxytocin (OT) release from the rat hypothalamo-neurohypophysial (H-N) system. An additional aim was to determine whether the possible response of oxytocinergic neurons to these peptides could be modified by melatonin through a cAMP-dependent mechanism. The results show that the highly selective GnRH agonist (i.e., [Des-Gly(10),d-His(Bzl)(6),Pro-NHEt(9)]-LHRH; Histrelin) stimulates the secretion of OT from an isolated rat H-N system. Melatonin significantly inhibited basal and histrelin-induced release of OT in vitro, and displayed no significant influence on OT release in the presence of GnRH or its antagonist. Addition of melatonin to a medium containing forskolin resulted in significant reduction of OT secretion from the H-N system. On the other hand, addition of forskolin to a medium containing both histrelin and melatonin did not further alter the inhibitory influence of melatonin on the histrelin-dependent secretion of OT in vitro. Intracerebroventricular (icv) infusion (experiment in vivo) of a GnRH antagonist resulted in substantial inhibition of OT release, thus revealing the stimulatory action of endogenous GnRH. In melatonin-treated animals, blood plasma OT levels were not changed in comparison to the vehicle. Our present data strongly suggests that activation of the GnRH receptor in the hypothalamus is involved in stimulation of OT secretion from the rat H-N system. It has also been shown, under experimental in vitro conditions, that melatonin fully suppresses the response of oxytocinergic neurons to the GnRH agonist - histrelin. The effect of melatonin on OT release is mediated by the cAMP-dependent mechanism, although other mechanisms of action are also possible.

[The influence of melatonin on human reproduction]. / Wplyw melatoniny na czynnosc gonad u czlowieka.

This paper reviews the possible participation of melatonin in the process of human reproduction. The results of several studies have shown the clear correlation between melatonin and gonadotropins and/or sexual steroids, which suggest that melatonin may be involved in the sexual maturation, ovulation or menopause. Decreased secretion of melatonin which coexists with increased fertility in the summer is specific for women living on the north hemisphere. Moreover, abnormal levels of melatonin in the blood are associated with several disorders of the hypothalamus-pituitary-gonads axis activity, i.e., precocious or delayed pubertas, hypogonadotrophic or hypergonadotrophic hypogonadism or amenorrhoea. Melatonin binding sites have been demonstrated in the central nervous system (mainly in the pars dystalis of the pituitary and hypothalamic suprachiasmatic nucleus) as well as in the reproductive organs, e.g., human granulosa cells, prostate and spermatozoa. Melatonin can, therefore, influence the gonadal function indirectly--via its effect on gonadotropin-releasing hormone and/or gonadotropins secretion. It may also act directly; several data show that melatonin can be synthesized in gonads.

[The effect of melatonin on prolactin, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) synthesis and secretion]. / Wplyw melatoniny na synteze i wydzielanie prolaktyny, hormonu luteinizujacego (LH) i folikulotropowego (FSH).

This paper reviews the data on the role of the pineal gland and melatonin in the regulation of hypothalamo-adenohypophysial system activity. The results of in vivo and in vitro experiments show that the effect of melatonin on prolactin, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) synthesis and secretion depends on the animal species, age, sex, the concentration of the hormone, and experimental conditions. Moreover, the melatonin-responsive pars tuberalis of the pituitary is an intermediary in the control of prolactin secretion, while the melatonin-binding sites of the mediobasal hypothalamus are involved in the gonadotropic response to melatonin.

[The role of the pineal gland and melatonin in the regulation of adenohypophysial hormone synthesis and secretion]. / Rola szyszynki oraz melatoniny w regulacji syntezy i wydzielania wybranych hormonów czesci gruczolowej przysadki.

This paper reviews findings accumulated on the role of the pineal gland and its hormone,melatonin, in the regulation of hypothalamo-adenohypophysial system activity. The effect of melatonin on somatotrophin (GH), thyreotrophin (TSH), and adrenocorticotrophin (ACTH) synthesis and secretion is considered. Results of in vivo and in vitro experiments show that the influence of melatonin on adenohypophysial activity depends on the animal species, age, sex, the concentration of the hormone, and the experimental conditions. Melatonin modifies adenohypophysial hormone synthesis and secretion either directly by influencing the secretory activity of the cells in the anterior pituitary or indirectly by influencing the hypothalamic neurons producing the respective neurohormones which stimulate or inhibit the release of adequate adenohypophysial hormones. Melatonin acts via specific membrane receptors which have been demonstrated in several brain areas, with high concentrations in the hypothalamic suprachiasmatic nucleus and the pars tuberalis of the pituitary. It may also act directly on a genome. Moreover, the response of the hypothalamo-adenohypophysial system to melatonin may depend on this hormone's influence on the metabolism of some neuromediators and/or neuromodulators in the central nervous system.

Vasopressin release from the rat hypothalamo-neurohypophysial system: effects of tachykinin NK-1 and NK-2 receptors agonists and antagonists.

OBJECTIVES: Present experiments were undertaken to study the influence of peptide NK-1 and NK-2 receptor agonists and antagonists as well as substance P and neurokinin A (the natural ligands for these tachykinin receptors) on vasopressin (AVP) secretion from the rat hypothalamo-neurohypophysial (HN) system in vitro. RESULTS: The results showed that both substance P and highly selective tachykinin NK-1 receptor agonist, i.e., [Sar9,Met(O2)11]-Substance P, enhanced significantly AVP secretion, while the NK-1 receptor antagonist (Tyr6,D-Phe7,D-His9)-Substance P (6-11)--sendide--was found to antagonize the substance P-induced hormone release from isolated rat HN system (all peptides at the concentration of 10(-7) M/L). The NK-2 receptor selective agonist (beta-Ala8)-Neurokinin A (4-10) was essentially inactive in modifying AVP release from the rat HN system in vitro, while neurokinin A (the natural ligand for this tachykinin receptor) was found to stimulate the AVP release; this effect of neurokinin A has been diminished by the NK-2 receptor antagonist (Tyr5,D-Trp(6,8,9),Lys-NH2(10))-Neurokinin A (4-10). CONCLUSION: The present data indicate a role for tachykinin NK-1 (and possibly for NK-2) receptors in tachykinin-mediated stimulation of AVP secretion from the rat HN system in vitro.

Melatonin inhibits the substance P-induced secretion of vasopressin and oxytocin from the rat hypothalamo-neurohypophysial system: in vitro studies.

The aim of the present investigations was to study the influence of substance P (a member of a family of peptides known as tachykinins) on basal and K(+)-evoked vasopressin (AVP) and oxytocin (OT) release from rat hypothalamo-neurohypophysial system in vitro as well as to determine whether this effect of substance P is sensitive to melatonin. The present results show that substance P stimulates basal AVP and OT release from isolated hypothalamo-neurohypophysial system, when used at the concentrations of 10(-6) and 10(-7)M/l. At the concentration of 10(-9)M/l, however, substance P was found to stimulate the in vitro secretion of AVP, but not that of OT. Melatonin diminished basal release of AVP; it also significantly inhibited the substance P-stimulated secretion of AVP and OT. K(+)-evoked release of the neurohypophysial hormones was not further modified by either substance P or melatonin. The present results show that the stimulatory effect of substance P on basal release of AVP and OT from rat hypothalamo-neurohypophysial system in vitro is sensitive to inhibitory influence of melatonin.

Septal vasopressin induced preservation of social recognition in rats was abolished by pinealectomy.

The role of intraseptal vasopressin (AVP) and the pineal gland in the modulation of social memory was investigated. For social recognition, male pinealectomised (Px) and sham-operated (SO) rats were confronted with juveniles for 4 min, and injected with either 200 pg AVP or 5 ng of its V1 receptor antagonist d(CH2)5Tyr(Me)AVP (AAVP) into the mediolateral septum. Re-exposure to the same and a different juvenile took place after 30 or 120 min. In SO rats, the social memory was facilitated after injection of AVP (120 min) and impaired after AAVP application (30 min). In Px rats, however, neither AVP nor AAVP administration influenced the social memory. Comparison between SO and Px control groups treated with artificial cerebrospinal fluid did not reveal any differences as to social recognition responses. After subcutaneous administration of 250 microg melatonin (substitution for pinealectomy) the social discrimination responses in Px rats after peptide application were influenced in a manner identical to that noted in SO animals. These findings suggest that the modulation of social memory by intraseptal AVP is dependent on an intact pineal function.

Effects of oxytocin and vasopressin on retrieval of passive avoidance response in melatonin-treated and/or pinealectomized male rats.

The role of the pineal gland and its hormone-melatonin-as to the impact of vasopressin (VP) and/or oxytocin (OT) on the regulation of behavior was studied, the passive avoidance task being chosen as an experimental model. The results showed that VP facilitated the avoidance latency during the first retention trial; after pinealectomy, however, VP was ineffective in this regard. Intraperitoneal application of OT was ineffective in modifying the passive avoidance latency when compared with respective saline-treated animals. Melatonin alone, when injected to shamoperated animals 30 min before behavioral experiment, did not affect the passive avoidance response in SA- or OT-treated rats, but blocked the VP-induced lengthening of the passive avoidance latency in the first retention trial. In pinealectomized and OT-treated animals the passive avoidance latency during the second retention trial was severely diminished by melatonin when compared to respective control. We conclude that: a) VP needs a regulated pineal function for developing short-term effects on the passive avoidance response and b) the effect of OT on the avoidance latency in pinealectomized rats develops after melatonin treatment as a long-term effect.