Short-term administration of melatonin or ghrelin on diabetic rats: effects on angiotensin II and vasopressin-induced uterine contractility.

The aim of the present study was to investigate the effects of Angiotensin II (Ang II) and Arginin-Vasopressin (AVP) on contractility of non-pregnant uterus in diabetic Wistar rats and to explore whether one-week administration of Melatonin (MLT) or Ghrelin (GHR) will change the response of diabetic uterine muscle to AngII and AVP. Uterine horns, prepared by the method of isolated tissues were investigated as well as glycemic profile, blood pressure and body weight. The research of smooth muscle contractions was made by a new method of analysis, characterizing in detail the various phases of the myometrial activity. Differences in the development of the peptide-mediated smooth muscle contractions depending on the phase of the estrous cycle were observed. Experimental diabetes had a pronounced negative effect on force and time-parameters of AngII and AVP-stimulated uterine contractions. Administration of GHR or MLT had a beneficial effect on the glycemic status of diabetic rats and partially improved the response of uterine preparations to the peptides. The application of MLT increased both force and time-parameters of Ang II-and AVP-stimulated uterine contractions while treatment with GHR increased power characteristics and shortened contraction and relaxation of the smooth muscle process.

Blood coagulation, fibrinolysis, and markers of endothelial dysfunction in systemic sclerosis.

OBJECTIVE: To evaluate the coagulative/fibrinolytic cascade and the circulating markers of the endothelial injury in systemic sclerosis (SSc). METHOD: Plasma was obtained from 29 patients with SSc and tested for thrombin-antithrombin (TAT), fragments 1+2 (F1+2), dermatansulphate (DS), thrombomodulin (TM), lipoprotein (a) [Lp(a)], von Willebrand factor (vWF), tissue type plasminogen activator (tPA), plasminogen activator inhibitor (PAI), D-dimers, intercellular adhesion molecole-1 (ICAM-1), vascular cell adhesion molecule (VCAM), and E-selectin. The data were correlated with lung (forced vital capacity, diffusing lung capacity for carbon monoxide, vital capacity) and skin (skin score) involvement. RESULTS: Coagulation was significantly activated (increase in F1+2, P <.001; TAT, P <.01; and Lp(a), P <.05). TM was not significantly different from controls. vWF was significantly increased (P <.01), and its supranormal multimers increased in more than 50% of patients. DS was significantly increased in diffuse cutaneous SSc (P <.01). Fibrinolysis was impaired as shown by reduced D-dimers (P <.01) and decreased levels of PAI (P < 0.01). The markers of endothelial injury were also significantly elevated. DS correlated significantly with forced vital capacity (P <.01) and forced vital capacity ratio (P <.01). CONCLUSION: Injury to the endothelium reduces endothelial function, as suggested by impairment of fibrinolysis and activation of the coagulative pathway. The loss of the balance between fibrinolysis and coagulation contributes to vessel engulfment with fibrin and breakdown of vessel patency. The increase of circulating DS suggests that this factor may be a new marker of endothelial injury.

Neurotensin modulation of acetylcholine and GABA release from the rat hippocampus: an in vivo microdialysis study.

The effects of neurotensin (NT) on the release of acetylcholine (ACh), aspartate (Asp), glutamate (Glu) and gamma-aminobutyric acid (GABA) from the hippocampus of freely moving rats were studied by transversal microdialysis. ACh was detected by High Performance Liquid Chromatography (HPLC) with electrochemical detection while GABA, glutamate and aspartate were measured using HPLC with fluorometric detection. Neurotensin (0.2 and 0.5 microM) administered locally through the microdialysis probe to the hippocampus produced a long-lasting and concentration-dependent increase in the basal extracellular levels of GABA and ACh but not of glutamate and aspartate. The increase in the extracellular levels of GABA and ACh produced by 0.5 microM neurotensin in the hippocampus reached a maximum of about 310% for GABA and 250% for ACh. This stimulant effect of NT was antagonized by the NT receptor antagonist SR 48692 (100 microg/kg, i.p.). Local infusion of tetrodotoxin (1 microM) decreased the basal release of ACh, GABA, Asp, Glu and prevented the 0.2 microM NT-induced increase in GABA and ACh release. The effect of NT on the release of ACh was blocked by the GABA(A) receptor antagonist bicuculline (2-10 microM). Our findings indicate for the first time that neurotensin plays a neuromodulatory role in the regulation of GABAergic and cholinergic neuronal activity in the hippocampus of awake and freely moving rats. The potentiating effects of neurotensin on GABA and ACh release in the hippocampus are probably mediated by (i) NT receptors located on GABAergic cell bodies and (ii) through GABA(A) receptors located on cholinergic nerve terminals.

Coordinated role of vasoactive intestinal peptide and nitric oxide in cardioprotection.

The present study sought to examine the interrelationship between nitric oxide (NO) and vasoactive intestinal peptide (VIP) in myocardial protection. Isolated rat hearts were perfused for 15 min with buffer only (Group I); 0.3 mM VIP (Group II); 3 mM L-arginine (a precursor of NO) (Group III); VIP and aminoguanidine (iNOS blocker) (Group IV); or L-arginine plus VIP 10-28 (VIP inhibitor) (Group V). Each heart was then made globally ischemic for 30 min followed by 2 h reperfusion. Both VIP and NO were found to provide cardioprotection during ischemia and reperfusion. However, the beneficial effects of VIP and NO were reduced by inhibition of NO and VIP, respectively, suggesting that cardioprotection by VIP is modulated by NO and vice versa. The results of this study suggested a coordinated regulation by cardioprotection by NO and VIP.

Cholinergic-nitrergic interactions in the guinea-pig gastric fundus.

The influence of nitric oxide (NO) on the spontaneous tone and on the contractile responses to electrical field stimulation or to exogenous acetylcholine (ACh) was studied. Circular strips from the guinea-pig gastric fundus were used. The NO-releasing compound sodium nitroprusside reduced the spontaneous tone while the NO-synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) increased it. The L-NAME-induced increase of the tone was antagonized by atropine or indomethacin, suggesting the involvement of cholinergic and prostaglandinergic pathways in this effect. L-NAME significantly potentiated the ACh (10(-8) to 10(-5) M)-induced contractions. L-NAME concentration-dependently potentiated the cholinergic contractions evoked by electrical field stimulation without affecting [3H]ACh overflow from [3H]choline-treated tissues. It is concluded that electrical field stimulation of gastric fundus muscle induces the release of endogenous nitrate which, in turn, functionally antagonizes cholinergic neurotransmission.

Effect of neurotensin on the canine gallbladder motility: in vivo and in vitro experiments.

Neurotensin (NT) (10(-8)-10(-6)) exerted a dose-dependent increase in the tone and release of [3H]ACh in the guinea-pig gallbladder muscle strips but was inefficient in the canine gallbladder muscle strips. However, in conscious dogs NT (2.5-20 ng/kg intravenously (i.v.)) dose-dependently increased the gallbladder pressure. Similar was the effect of CCK8 (1-10 ng/kg i.v.) and carbachol (0.5-2 micrograms/kg i.v.). The NT- or CCK8-induced gallbladder pressure was inhibited by atropine (10-50 micrograms/kg i.v.) or hexamethonium (0.5-3 mg/kg i.v.). Somatostatin (1-2 micrograms/kg i.v.) or VIP (0.5-1 microgram/kg i.v.) also reduced or even abolished the NT- or CCK8-induced gallbladder pressure. The NT-induced increase of the tone of guinea-pig gallbladder preparations was accompanied by an increase of [3H]ACh release, suggesting the involvement of cholinergic innervation.

Effect of somatostatin on the canine gallbladder motility.

Somatostatin (SOM) at doses up to 1 microgram was not effective on the motility of canine and guinea pig gallbladder smooth muscle preparations in vitro. When the preparations were contracted by field electrical stimulation (0.7 ms, 40 Hz) the cholecystokinin octapeptide (CCK OP) enhanced these contractions while SOM inhibited them. These effects were accompanied, respectively, by an increase or a decrease in [3H] acetylcholine (ACh) release in the intrinsic cholinergic nerve terminals. SOM (0.5 to 2 micrograms/kg i.v.) inhibited the spontaneous and the CCK OP-activated gallbladder pressure in conscious dogs. The effect of atropine (10-50 micrograms/kg) was similar to that of SOM when injected intravenously in conscious dogs. It is suggested that the inhibitory effect of SOM on gallbladder pressure in conscious dogs is probably mediated by a decrease in ACh release by cholinergic neurons.

Influence of steroids on complement and cytokine generation after cardiopulmonary bypass.

BACKGROUND: It is recognized that the inflammatory mediators complement and cytokines are generated during cardiopulmonary bypass as an endogenous response to extracorporeal circulation. METHODS: Nineteen randomized patients (10 steroid/9 nonsteroid) entered an institutional review board-approved protocol to measure complement and interleukin level generation before and after elective coronary revascularization. The steroid regimen involved 1 g of methylprednisolone sodium succinate intravenously before bypass and 4 mg of dexamethasone every 6 hours for four doses during the first 24 hours of recovery. Complement and interleukin levels were measured before bypass, immediately after bypass, and at 24, 48 and 72 hours of recovery. RESULTS: In the nonsteroid group, there was a significant elevation in all inflammatory mediators relative to the steroid group. The predominant changes occurred at 24 hours after operation. CONCLUSIONS: Steroids produced a dramatic reduction in complement and interleukin levels. The number of patients was clearly too small to document a clinical consequence of steroid administration.

NO synthesis inhibition decreases cortical ACh release and impairs retention of a conditioned response.

We investigated in rats the effect N(G)-nitro-L-arginine methyl ester (L-NAME) on retention of a passive avoidance response, and cortical ACh release monitored using the microdialysis technique. Post-training administration of L-NAME impaired 24 h retention of a passive avoidance and decreased cortical ACh release. Both effects of L-NAME were reversed by L-Arg. These results suggest that nitric oxide is involved in retention of the passive avoidance response through the modulation of the forebrain cholinergic system.

Vasoactive intestinal peptide protects guinea-pig detrusor nerves from anoxia/glucopenia injury.

Vasoactive intestinal peptide (VIP) was tested for its capability to protect the intrinsic nerves of guinea-pig urinary bladder from damage due to anoxia/glucopenia and reperfusion. Guinea-pig detrusor strips were mounted for tension recording in small organ baths and the nerves were subjected to electric field stimulation. VIP (0.3 microM) improved significantly the response of strips to electrical field stimulation either during anoxia/glucopenia or thereafter during reperfusion, as compared to untreated tissues. The antioxidant activity of VIP assessed as its capability to scavenge peroxyl radicals during linoleic acid oxidation corresponded to 6.42+/-0.13 pIC(50) M, i.e. close to the concentration proved to protect strips against the anoxic--glucopenic and reperfusion damage.

Mechanical response to electrical field stimulation of rat, guinea-pig, monkey and human detrusor muscle: a comparative study.

The aim of the present investigation was to compare mechanical responses to electrical field stimulation (EFS), as well as cholinergic and non-adrenergic, noncholinergic (NANC) neurotransmission in guinea-pig, rat, monkey and human detrusor muscle strips. Responses to EFS (0.05, 0.5 and 1 ms pulse duration, 50 V, 1-15 Hz) of guinea-pig, rat, monkey and human detrusor muscle strips were recorded isometrically before and after blockade of muscarinic receptors and/or P2-purinoreceptors, as well as after desensitisation of P2-purinoceptors or blockade of the nerve impulse propagation. Single pulses of 0.05 ms duration elicited responses, in either guinea-pig or rat detrusor strips, which were abolished by tetrodotoxin (TTX), thus suggesting their neurogenic nature. In monkey and human detrusor strips, however, the same single pulses were not sufficient to generate contractile responses. The response of either rat or guinea-pig strips to single pulses of 0.5 ms and 1 ms duration was mainly myogenic in nature. While in rat and guinea-pig strips the neurogenic response was only partly reduced in the presence of atropine, in monkey and human strips it was abolished. In the presence of atropine, while suramin only partially reduced the EFS response either in rat or guinea-pig detrusor strips, a complete alpha,beta-methyleneATP-sensitive response was evident in guinea-pig detrusor strips. This suggests the involvement of other transmitter(s) beyond ATP in the NANC response of rat detrusor strips.

Neuroprotection afforded by some hindered phenols and alpha-tocopherol in guinea-pig detrusor strips subjected to anoxia-glucopenia and reperfusion-like conditions.

2-t-butyl-4-methoxyphenol (BHA), 3,5-di-t-butyl-hydroxyanisole (DTBHA), 2,6-diisopropylphenol (propofol), alpha-tocopherol (alpha-TOC) and two newly synthesised analogues of BHA, namely 1-O-(4-hydroxy-3-t-butyl)phenyl-2,3,4,6-tetra-O-acetyl-beta-D-glucopyranose (beta-TAG) and 1-O-(4-hydroxy-3-t-butyl)phenyl-beta-D-glucopyranose (beta-GLU), were tested for their capability to protect the intrinsic nerves of guinea-pig urinary bladder from damage due to anoxia-glucopenia and re-exposure to glucose and O2. Guinea-pig detrusor strips were mounted for tension recording in small organ baths, superfused with warmed Krebs solution and the nerves stimulated electrically either under control or ischaemia-like (anoxia-glucopenia) and reperfusion-like conditions (normal medium re-superfusion). The Ca2+ antagonist activity of the compounds was assessed by their effect on the contraction of detrusor strips induced by 60 mM K+ Krebs solution in the presence of either 0.5 mM or 5 mM Ca2+. The antioxidant activity was illustrated by the ability of the compounds to scavenge peroxyl radicals generated by linoleic acid oxidation. All the compounds, except beta-GLU and alpha-TOC, inhibited in a concentration-dependent manner K+-induced contractions of detrusor muscles, the inhibition being inversely related to the Ca2+ concentration of the perfusion solution; moreover, they exhibited a marked antiperoxidant activity with pIC50 values decreasing in the order: DTBHA > alpha-TOC > BHA > beta-TAG > propofol > beta-GLU. alpha-TOC, BHA, DTBHA and beta-TAG improved significantly the response of the strips to electrical field stimulation either during the anoxia-glucopenia phase or thereafter when recovering during reperfusion, as compared to untreated tissues. The neuroprotection afforded by the phenol derivatives as well as by alpha-TOC was positively correlated to their antioxidant activity, but not to their Ca2+ antagonist activity.

Cholinergic mechanism in cholecystokinin action on gall bladder motility.

The participation of cholinergic mechanisms in cholecystokinin octapeptide (CCKOP) action on canine gall bladder was studied in vivo and in vitro, using three different experimental conditions. In vitro the responses of canine gall bladder smooth muscle to CCKOP (0.01 to 10 nm) were insensitive to atropine (1 to 10 microM) and tetrodotoxin (3 microM). When gall bladder muscle preparations were contracted by field electrical stimulation (0.7 ms, 40 Hz) CCKOP (0.001 to 0.1 nM) enhanced these contractions while atropine (1 microM) abolished them. This suggests that CCKOP is able to influence acetylcholine (ACH)-release from intrinsic cholinergic nerve terminals. In vivo the responses of canine gall bladder smooth muscle to CCKOP (1 to 10 ng/kg i.v.) were reduced and even abolished by atropine (10 to 50 micrograms/kg i.v.) and hexamethonium (0.5 to 3 mg/kg i.v.). The results suggest the participation of at least two mechanisms in CCKOP action on canine gall bladder motility: a direct action on smooth muscle cells, insensitive to atropine or tetrodotoxin, and an indirect action, which is dependent on pre- and post ganglionic cholinergic pathways.

Participation of extracellular Ca(2+) or ghrelin in peptide-mediated contraction of strips from rat urinary bladder.

Many neuropeptides, like angiotensins and vasopressin, are involved in the regulation of urinary bladder contractile activity. They activate the inositol-triphosphate signal pathway and increase intracellular calcium concentration. To determine the effect of ghrelin and extracellular calcium on the amplitude and force of angiotensin and vasopressin-mediated contractions of detrusor, we used strips from urinary bladder of Wistar rats. The obtained preparations were stimulated with Angiotensin II (Ang II) and arginine-vasopressin (AVP), independently and in combination with CaCl(2) or ghrelin. The simultaneous application of peptide and CaCl(2) increased the amplitude and the integral force (AUC) of muscle contraction for both neuropeptides. If ghrelin was applied to the preparation 30min prior to application of Ang II, it prevented the development of typical Ang II-mediated contraction. Ghrelin did not affect the amplitude of AVP-mediated contraction, but significantly lowered its integral force. Our experimental data indicate that the increase of calcium in the extracellular fluid possesses a synergistic effect on the neuropeptide-mediated contraction. The effects of ghrelin on Ang II- and AVP-mediated contractions allow us to express the assumption that the urinary bladder cells probably have ghrelin receptors which do not activate phospholipase C signal pathway.

The effect of vasoactive intestinal polypeptide (VIP) on the canine gallbladder motility.

1. VIP at doses of 10(-9) to 10(-8) M was ineffective and at doses of 5 x 10(-8) to 10(-7) M exerted a slight inhibitory effect on the tone of the canine gallbladder muscle strip. However, VIP (0.1-1 micrograms/kg) injected intravenously (i.v.) in conscious dogs dose-dependently decreased the gallbladder pressure. 2. VIP did not influence significantly the acetylcholine (ACh)- or carbachol- induced contractions of canine gallbladder under in vitro or in vivo conditions, but it decreased the electrically-induced, atropine-sensitive contractions of gallbladder muscle strips. 3. VIP (5 x 10(-9) to 5 x 10(-8) M) did not influence significantly the dose-response curve for cholecystokinin octapeptide (CCK OP) of canine and guinea-pig gallbladder muscle strips. VIP injected i.v. (0.1-0.5 micrograms/kg) in conscious dogs greatly decreased the CCK OP-induced gallbladder pressure.

Effect of vasoactive intestinal peptide (VIP) on the mechanical activity and [3H] acetylcholine release in guinea-pig gastric muscle.

Muscle strips, 30 x 3 mm, were cut out in circular direction from the fundus region of the stomach. Mechanical activity was recorded by means of mechanoelectrical force transducers. Vasoactive intestinal peptide (VIP) at concentrations of 10(-9) to 10(-7) M caused a dose-dependent relaxation of muscle strips which was insensitive to tetrodotoxin. Field electrical stimulation (10 Hz, 1 ms, supramaximal voltage) produced contractions, which were blocked by atropine. VIP (10(-9) to 10(-7) M) dose-dependently decreased the amplitude of the electrically-induced contractions. VIP dose-dependently reduced the electrically-stimulated [3H] acetylcholine release. It is suggested that at least two different mechanisms are involved in the VIP-induced relaxation of the gastric muscle: a direct action on the smooth muscle and an indirect action mediated through cholinergic innervation.

Effects of cholecystokinine on the gallbladder motility: interaction with somatostatin and vasoactive intestinal peptide.

Recent years have seen an increase in the information concerning the mechanisms of action of brain-gut neuropeptides (cholecystokinin (CCK), vasoactive intestinal peptide (VIP), somatostatin (SS)) on the biliary tract motility. This article is intended to extend our knowledge of the problem and based on our recent studies. Researchers and students interested in an historical overview of the subject, as well as in the information on the physiology and pharmacology of biliary smooth muscle are referred to earlier reviews (Ryan, 1981, 1987). The article focuses on the involvement of cholinergic mechanisms in the action of CCK, SOM and VIP on the gallbladder motility under in vivo and in vitro conditions. Some species differences in the responses of the gallbladder to CCK, VIP and ACh have also been described. Furthermore, new data about the interactions between CCK, SOM and VIP in the regulation of the gallbladder motility are presented.

Neuropeptides of the cholecystokinin group: effects and mechanisms of action on the gastro-intestinal and gall bladder motility.

The neuropeptides of the cholecystokinin (CCK) group belong to the substances usually referred to as "brain-gut" neuropeptides. They are synthesized in neurons of the central nervous system, in the peripheral and in the autonomous nervous systems, in endocrine cells (types "I", "K" and "A"), as well as in the enteric nervous system of the gastro-intestinal tract and of the pancreas. The CCK-group peptides realize their effects via several different mechanisms (Fig. 1): endocrine or neuroendocrine (classic hormonal mechanism)--the peptide, released by the endocrine cell or by the nerve terminal, is carried by the circulation to the remote target organs; paracrine or neuroparacrine--the peptide, released in the intercellular space, reaches the target effector cells via diffusion. Similarly to the classic neurotransmitters, CCK and its analogues could play a neurotransmitter role, also modulating the release of acetylcholine (ACh) and of other neurotransmitters in enteric and CNS neurons. In the present review article some smooth-muscle and neuromodulatory effects of CCK are described and compared to the results of the authors' studies on the problem.

Glutamatergic modulation of cortical acetylcholine release in the rat: a combined in vivo microdialysis, retrograde tracing and immunohistochemical study.

The microdialysis technique with one or two probes was used to investigate the modulation of cortically projecting cholinergic neurons by glutamatergic input in the rat in vivo. Male albino Wistar rats (250-300 g) were used. Under chloral hydrate anaesthesia microdialysis membranes were positioned in the parietal cortex, nucleus basalis magnocellularis (NBM) or medial septum. Acetylcholine was assayed using high-performance liquid chromatography (HPLC) with electrochemical detection while GABA was detected using HPLC with fluorimetric detection after derivatization of the amino acid with o-phthalaldehyde. Septo-cortical neurons were retrogradely labelled with fluoro-gold. Double labelling with choline acetyltransferase (ChAT) immunoreactivity was performed to identify these neurons. Our main findings were that: (i) i.c.v. administration of the NMDA antagonist 3-((R)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP, 1-5 nmol) increased cortical acetylcholine outflow; (ii) local administration of CPP (100 microM) to the cortex had no effect on cortical acetylcholine outflow; (iii) local administration of CPP (100 microM) to the NBM decreased cortical acetylcholine outflow; (iv) local administration of CPP (100-200 microM) to the septum increased cortical GABA and acetylcholine outflow; (v) administration of muscimol to the septum prevented the effect of CPP on cortical acetylcholine outflow; (vi) retrograde tracing with fluoro-gold labelled cell bodies in the medial septum; (vii) septal fluoro-gold-positive neurons were not ChAT-immunoreactive. Our in vivo neurochemical results, in combination with retrograde tracing and immunohistochemistry, indicate that the cortically projecting cholinergic system is indirectly regulated by a glutamatergic input via a polysynaptic GABAergic circuitry located in the septum.

Effects of bombesin on the canine gallbladder motility: in vivo and in vitro experiments.

The effects of bombesin (BM) on the canine gallbladder motility was studied under two different experimental conditions: (i) in conscious dogs with a balloon chronically implanted into the gallbladder lumen where intragallbladder pressure was recorded in mm Hg by means of a pressure transducer, and (ii) in smooth muscle preparations isolated from different regions of the gallbladder where the contractions were recorded isometrically by means of mechanoelectrical transducers. Similar to CCK8 bolus injection of. BM i.v. increased the gallbladder pressure in a dose-dependent manner. The response was characterized by a slow increase of the tone and a gradual restoration (in 4 to 8 min) of the background activity. The threshold dose and the maximum dose were 30 ng/kg and 100 ng/kg for BM and 1 ng/kg and 10 ng/kg for CCK8, respectively. Both atropin (10 to 50 micrograms/kg) and hexamethonium (0.5 to 3 mg/kg) injected i.v. 5 to 10 min before BM strongly reduced or even abolished the gallbladder response to BM. Somatostatin (1 to 2 micrograms/kg) and VIP (0.5 to 1 microgram/kg) injected 3 to 5 min before BM also exerted a strong inhibitory effect on the canine gallbladder response to BM. However BM (up to 10(-6) M) had no effect on the spontaneous or electrically-induced contractions of the canine gallbladder smooth muscle preparations. The results suggest the involvement of prejunctional cholinergic-, somatostatinergic- and VIP-ergic pathways in the bombesin-induced increase of the gallbladder pressure of conscious dogs.