New Horizons in the Etiopathogenesis and Non-Invasive Diagnosis of Endometriosis.

Endometriosis is one of the most common gynecological inflammatory diseases, occurring in adolescents and women in the reproductive age group and leading to infertility. The precise etiopathogenesis of endometriosis is unknown, but several theories concerning the phenomena involved in its development have been proposed. Beside classic retrograde menstruation, these include lymphatic and vascular metastases, iatrogenic direct implantation, coelomic metaplasia, embryonic remnants and mesenchymal cell differentiation or induction; the persistence of a form of embryonic endometriosis may also be involved, as well as the theory of the possible role of endometrial stem/progenitor cells. This paper deals with other risk factors which may be potentially involved in the etiopathogenesis of endometriosis, including the immune, inflammatory, endocrine, genetic, anatomical and environmental factors. At present, endometriosis can only be diagnosed with surgery, where laparoscopy is considered a gold standard. Therefore, there is an urgent need for a test allowing to detect non-invasive molecular biomarkers to identify the symptoms of endometriosis early on in disease development. A thorough understanding of the etiopathogenesis of endometriosis is essential toward the development of novel diagnostic assays and effective treatments of the disease.

A new face of endocannabinoids in pharmacotherapy. Part I: protective role of endocannabinoids in hypertension and myocardial infarction.

Cannabinoids are compounds which were first isolated from the Cannabis sativa plant. For thousands of years they have been used for treatment of numerous diseases. Currently, synthetic cannabinoids and endocannabinoids are also known. Cannabinoid receptors, endocannabinoids and the enzymes that catalyze their synthesis and degradation constitute the endocannabinoid system which plays an important role in functioning of the cardiovascular system. The results obtained to date suggest the involvement of endocannabinoids in the pathology of many cardiovascular diseases, including myocardial infarction, hypertension and hypotension associated with hemorrhagic, endotoxic, and cardiogenic shock. Cardioprotective effect and dilation of coronary vessels induced by endocannabinoids deserve special attention. It cannot be excluded now that in the future our better understanding of cannabinoid system will allow to develop new strategies for treatment of cardiovascular diseases.

A new face of endocannabinoids in pharmacotherapy. Part II: role of endocannabinoids in inflammation-derived cardiovaascular diseases.

Endocannabinoids play an important role in cardiovascular diseases caused by inflammatory disorders. Endocannabinoids are endogenous bioactive lipids that activate cannabinoid receptors and together with enzymes responsible for their synthesis and degradation constitute endocannabinoid system. The results obtained to date suggest the involvement of endocannabinoids in the pathology of many cardiovascular diseases associated with inflammation, such as atherosclerosis, restenosis, chemotherapy-induced myocardial injury, diabetic and hepatic cirrhosis cardiomyopathy. Our better understanding of cannabinoid system may result in the development of new strategies for the treatment of such disorders.

Transient receptor potential vanilloid 4 blockade protects against experimental colitis in mice: a new strategy for inflammatory bowel diseases treatment?

Recent reports suggested that the activation of Transient Receptor Potential Vanilloid 4 (TRPV4) receptors in the gastrointestinal tract has pro-inflammatory effects. In this study, we demonstrated for the first time that TRPV4 mRNA expression is up-regulated in patients with inflammatory bowel diseases (IBD). Furthermore, selective blockade of TRPV4 in the 2,4,6-trinitrobenzenesulfonic acid animal model alleviates colitis and pain associated with the intestinal inflammation. Our study indicates that TRPV4 may play a role in mechanisms of defense in intestinal inflammation and that TRPV4 may be an attractive target for future systemic or topic anti-inflammatory treatment in patients with IBD.

The influence of opioid peptides on matrix metalloproteinase-9 and urokinase plasminogen activator expression in three cancer cell lines.

Matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA) regulate proteolysis of the extracellular matrix (ECM) and as a consequence are involved in a number of physiological and pathological states, including cancer. A crucial feature of cancer progression and metastasis is the disruption of the ECM and spreading of proliferating cancer cells. Over-expression of MMPs and uPA is common for most types of cancers and correlates well with the adverse prognosis. Compounds able to modulate the activity of these proteolytic enzymes may become important agents in cancer therapy. In the present study, we examined the effect of the mu-opioid receptor selective peptide, morphiceptin, and its two synthetic analogs on mRNA and protein levels of MMP-9 and uPA in three human cancer cell lines: MCF-7, HT-29, and SH-SY5Y. Our findings indicate that in all three cell lines morphiceptin and its analogs attenuated MMP-9 expression and secretion and that this effect is not mediated by opioid receptors but is under control of the nitric oxide system. On the other hand, tested opioids up-regulated uPA levels through a mechanism that involved opioid-receptors. Different pathways by which opioid peptides exert their actionin cancer cells can explain their contradictory influence on the level of cancer markers.

Differential effects of salvinorin A on endotoxin-induced hypermotility and neurogenic ion transport in mouse ileum.

BACKGROUND: Salvinorin A (SA) is the principal active ingredient of Salvia divinorum, with an established inhibitory action on gastrointestinal (GI) transit and colonic ion transport in mice. Under normal conditions, the effects of SA are mediated by kappa opioid (KOR) and cannabinoid (CB1 and CB2) receptors. However, the role of SA in pathophysiological conditions remains unresolved. The aim of this study was to characterize the in vitro and in vivo effects of SA on mouse ileum after endotoxin challenge. METHODS: Changes in GI motility were studied in vitro, using smooth muscle preparations from the mouse ileum. In vivo, the fecal pellet output and small intestinal fluid content were measured. Neurogenic ion transport and intestinal permeability were examined using Ussing chambers. In addition, Western blot analysis of mucosa was performed and plasma nitrite/nitrate levels were determined. KEY RESULTS: Salvinorin A inhibited endotoxin-induced ileal hypercontractility via KOR, CB1, and CB2 receptors. Neurogenic ion transport, which was significantly reduced after endotoxin challenge, was normalized by SA through a nitric oxide synthase (NOS)-dependent mechanism. Western blot analysis and plasma nitrite/nitrate level quantitation confirmed the involvement of NOS in the regulatory action of SA. CONCLUSIONS & INFERENCES: This is the first report showing differential effects of SA on motor and secretory activity in mouse GI during endotoxemia. The outcomes of our study imply possible novel applications of SA and its analogs in the treatment of GI disorders.

Development of opioid peptide analogs for pain relief.

Opioid receptors and opioid peptides constitute the endogenous opioid system. The most relevant function of the opioid system seems to be the inhibitory modulation of nociceptive information at supraspinal, spinal and peripheral sites, although it is also implicated in the modulation of many other processes in the body. Centrally acting plant opiates, such as morphine, are the most frequently used analgesics for the relief of severe pain, even though their undesired side-effects are serious limitation to their usefulness. Opioid peptides have the potential to be pharmaceutical agents for the treatment of pain, devoid of side-effects accompanying morphine. Unfortunately, peptides are generally hydrophilic compounds that will not enter the central nervous system via passive diffusion, due to the existence of the blood-brain barrier. Peptides are also easily degraded by proteolytic enzymes which further reduces their therapeutic value. Therefore, the design of peptide analogs based on the sequence of endogenous opioid peptides must be focused on increasing bioavailability and enhancing brain uptake.

Salvinorin A inhibits colonic transit and neurogenic ion transport in mice by activating kappa-opioid and cannabinoid receptors.

The major active ingredient of the plant Salvia divinorum, salvinorin A (SA) has been used to treat gastrointestinal (GI) symptoms. As the action of SA on the regulation of colonic function is unknown, our aim was to examine the effects of SA on mouse colonic motility and secretion in vitro and in vivo. The effects of SA on GI motility were studied using isolated preparations of colon, which were compared with preparations from stomach and ileum. Colonic epithelial ion transport was evaluated using Ussing chambers. Additionally, we studied GI motility in vivo by measuring colonic propulsion, gastric emptying, and upper GI transit. Salvinorin A inhibited contractions of the mouse colon, stomach, and ileum in vitro, prolonged colonic propulsion and slowed upper GI transit in vivo. Salvinorin A had no effect on gastric emptying in vivo. Salvinorin A reduced veratridine-, but not forskolin-induced epithelial ion transport. The effects of SA on colonic motility in vitro were mediated by kappa-opioid receptors (KORs) and cannabinoid (CB) receptors, as they were inhibited by the antagonists nor-binaltorphimine (KOR), AM 251 (CB(1) receptor) and AM 630 (CB(2) receptor). However, in the colon in vivo, the effects were largely mediated by KORs. The effects of SA on veratridine-mediated epithelial ion transport were inhibited by nor-binaltorphimine and AM 630. Salvinorin A slows colonic motility in vitro and in vivo and influences neurogenic ion transport. Due to its specific regional action, SA or its derivatives may be useful drugs in the treatment of lower GI disorders associated with increased GI transit and diarrhoea.

Interactions of galanin with endomorphin-2, vasopressin and oxytocin in nociceptive modulation of the trigemino-hypoglossal reflex in rats.

Galanin (GAL) is suggested to be a neuropeptide involved in pain transmission. In this study we tried to determine, whether the increase of GAL concentration in brain cells affects impulse transmission between the motor centers localized in the vicinity of the third and fourth cerebral ventricles. The experiments were carried out on rats under chloralose anesthesia. The study objectives were realized using the method allowing to record the amplitude of evoked tongue jerks (ETJ) in response to noxious tooth pulp stimulation during the perfusion of the cerebral ventricles with solutions containing tested compounds. Perfusion of the cerebral ventricles with GAL concentration-dependently inhibited the ETJ amplitude. The antinociceptive effect of GAL was blocked by a galanin receptor antagonist, galantide (GLT) and by opioid antagonists: non-selective naloxone (Nal) and micro-selective beta-funaltrexamine (beta-FNA). In contrast, a delta-opioid receptor antagonist, naltrindole (NTI) or the kappa-opioid receptor antagonist, nor-binaltrophimine (nor-BNI) did not inhibit the effect of GAL. The antinociceptive effect of GAL was more pronounced when GAL was perfused in combination with other neuropeptides/neurohormones, such as endomorphin-2 (EM-2), vasopressin (AVP) and oxytocin (OT). The present results demonstrate that in the orofacial area analgesic activity is modulated by GAL, OT and AVP and that EM-2-induced antinociception involves GAL.

Effect of galanin on substance P- and vasoactive intestinal polypeptide-induced nociceptive trigemino-hypoglossal reflex in rats.

Substance P (SP), vasoactive intestinal polypeptide (VIP) and galanin (GAL), present in primary sensory neurons, are involved in transmission of nociceptive signaling from the peripheral to central nervous system. In this study we investigated the effect of GAL on SP-induced or VIP-induced evoked tongue jerks (ETJ) in response to noxious tooth pulp stimulation during perfusion of the cerebral ventricles with SP or VIP solutions. The experiments were carried out on rats under chloralose anesthesia. It was shown that both, SP and VIP, perfused through the cerebral ventricles enhanced the ETJ amplitude as compared with control, but the effect produced by SP was stronger. The intracerebroventricular perfusion of GAL 5 minutes before SP caused a dose-dependent inhibition of SP-induced ETJ, whereas GAL perfused through the cerebral ventricles 5 minutes before VIP did not reduce the excitatory effect of VIP on ETJ. These results indicate that the antinociceptive effect of GAL perfused through the cerebral ventricles, tested on the trigemino-hypoglossal reflex in rats, is specifically mediated by the SP-ergic system.

Effect of tooth pulp stimulation on oxytocin and vasopressin release into the cerebrospinal fluid and fluid perfusing the cerebral ventricles in rats.

OBJECTIVE: Stress- and pain-related stimuli cause a release of vasopressin (AVP) and oxytocin (OT) into the cerebrospinal fluid (CSF) and extracellular fluid of the brain in various animal species. The aim of the study was to investigate the effect of stimulation of the nociceptive afferent terminals in the tooth pulp on the release of AVP and OT into CSF in rats under chloralose anesthesia. METHODS: Cerebrospinal fluid was collected from the cerebellomedullary cistern and then 30-minute perfusions of the lateral cerebral ventricles with artificial cerebrospinal fluid (aCSF) were carried out. The perfusate was collected from the cerebellomedullary cistern at rest (control), during electric stimulation of the tooth pulp which induced nociceptive trigemino-hypoglossal reflex, and after stimulation. In the collected CSF and aCSF perfusates, AVP-like immunoreactivity (AVPLI) and OT-like immunoreactivity (OT-LI) were determined by radioimmunoassay (RIA). RESULTS: The concentrations of AVP-LI and OT-LI in CSF were found to reach 21 pg/ml and 67 pg/ml, respectively. Electric tooth pulp stimulation exerted no effect on AVP and OT release into the fluid perfusing the cerebral ventricles during stimulation. CONCLUSION: It was found that noxious stimulus from the tooth pulp is not a factor affecting significantly AVP and OT release into CSF.

Novel endomorphin-2 analogs with mu-opioid receptor antagonist activity.

A series of position 4-substituted endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) analogs containing 3-(1-naphthyl)-alanine (1-Nal) or 3-(2-naphthyl)-alanine (2-Nal) in L- or D-configuration, was synthesized. The opioid activity profiles of these peptides were determined in the mu-opioid receptor representative binding assay and in the Guinea-Pig Ileum assay/Mouse Vas Deferens assay (GPI/MVD) bioassays in vitro, as well as in the mouse hot-plate test of analgesia in vivo. In the binding assay the affinity of all new analogs for the mu-opioid receptor was reduced compared with endomorphin-2. The two most potent analogs were [D-1-Nal(4)]- and [D-2-Nal4]endomorphin-2, with IC50 values 14 +/- 1.25 and 19 +/- 2.1 nM, respectively, compared with 1.9 +/- 0.21 nM for endomorphin-2. In the GPI assay these analogs were found to be weak antagonists and they were inactive in the MVD assay. The in vitro GPI assay results were in agreement with those obtained in the in vivo hot-plate test. Antinociception induced by endomorphin-2 was reversed by concomitant intracerebroventricula (i.c.v.) administration of [D-1-Nal4]- and [D-2-Nal4]-endomorphin-2, indicating that these analogs were mu-opioid antagonists. Their antagonist activity was compared with that of naloxone. At a dose 5 microg per animal naloxone almost completely inhibited antinociceptive action of endomorphin-2, while [D-1-Nal4]endomorphin-2 in about 46%.

Binding of endomorphin-2 to mu-opioid receptors in experimental mouse mammary adenocarcinoma.

Endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) binds with high affinity and selectivity to the mu-opioid receptor. In the present study, [125I]endomorphin-2 has been used to characterize mu-opioid-binding sites on transplantable mouse mammary adenocarcinoma cells. Cold saturation experiments performed with [125I]endomorphin-2 (1 nM) show biphasic binding curves in Scatchard coordinates. One component represents high affinity and low capacity (K(d) = 18.79 +/- 1.13 nM, B(max) = 635 +/- 24 fmol/mg protein) and the other shows low affinity and higher capacity (K(d) = 7.67 +/- 0.81 microM, B(max) = 157 +/- 13 pmol/mg protein) binding sites. The rank order of agonists competing for the [125I]endomorphin-2 binding site was [d-1-Nal3]morphiceptin > endomorphin-2 >> [d-Phe3]morphiceptin > morphiceptin > [d-1-Nal3]endomorphin-2, indicating binding of these peptides to mu-opioid receptors. The uptake of 131I-labeled peptides administered intraperitoneally to tumor-bearing mice was also investigated. The highest accumulation in the tumor was observed for [d-1-Nal3)morphiceptin, which reached the value of 8.19 +/- 1.14% dose/g tissue.

Uptake of radiolabeled morphiceptin and its analogs by experimental mammary adenocarcinoma: in vitro and in vivo studies.

Morphiceptin (Tyr-Pro-Phe-Pro-NH(2)) and its analogs modified at position 3: [D-Phe(3)]morphiceptin, [D-ClPhe(3)]morphiceptin and [D-Cl(2)Phe(3)]morphiceptin were synthesized and labeled with [(125)I] or [(131)I]. Their binding to membranes isolated from experimental adenocarcinoma was examined in vitro with the use of a cross-linking assay followed by the Western blot technique. The radioactive complex had molecular weight of about 65 kDa and was detectable by anti-mu-opioid receptor polyclonal antibody. Expression of the mu-opioid receptor in mouse mammary adenocarcinoma was confirmed by reverse transcriptase-polymerase chain reaction. The binding studies showed the highest affinity and capacity for [D-Phe(3)]morphiceptin (K(d) 0.39 and B(max) 1112) and [D-ClPhe(3)]morphiceptin (K(d) 1.8 and B(max) 220). Morphiceptin and its D-Cl(2)Phe analog had significantly lower B(max) values (131 and 83, respectively). Biodistribution experiments in tumor-bearing C3H/Bi mice with the use of the (131)I-labeled peptides confirmed the results of our in vitro studies. The highest accumulation of radioactive peptides in the tumor tissue was also found for peptides with D-Phe and D-ClPhe.

Synthesis of novel morphiceptin analogues modified in position 3 and their binding to mu-opioid receptors in experimental mammary adenocarcinoma.

Binding of the (125)I-labeled mu-opioid receptor selective ligands, morphiceptin (Tyr-Pro-Phe-Pro-NH(2)) and [D-Phe(3)]morphiceptin, to membranes isolated from experimental mouse mammary adenocarcinoma was examined in vitro using a cross-linking assay followed by a Western blot technique. The radioactive complex had a molecular weight of about 65 kDa and was detectable by anti-mu-opioid receptor antibody, indicating the presence of mu-opioid receptors in tumor membranes. A series of new morphiceptin analogues, modified at the pharmacophoric position 3, was synthesized in order to find the correlation between the lipophilicity, electronic and steric properties of the amino acid in this position and the in vitro affinity of new analogues for mu-opioid receptors on mouse brain and tumor membranes. In in vivo studies the uptake of (131)I-labeled analogues by experimental mammary adenocarcinoma was estimated. The highest affinity for mu-opioid receptors in both, in vitro and in vivo experiments was observed for [D-Phe(3)]morphiceptin and [D-ClPhe(3)]-morphiceptin.

Effect of cerebral ventricles perfusion with morphiceptin and Met-enkephalin on trigemino-hypoglossal reflex in rats.

Opioids administered by intracerebroventricular injections produce analgesic responses in rats. The present study was undertaken to investigate the effects of a highly selective mu-opioid receptor ligand morphiceptin on trigemino-hypoglossal reflex in rats. The analgesic effect of morphiceptin was compared with another opioid peptide, Met-enkephalin. With the experimental settings used in this study, we have demonstrated that both morphiceptin and Met-enkephalin show significant dose-dependent analgesic effects after i.c.v. administration in rats as assayed by trigemino-hypoglossal reflex test. The antinociceptive response to Met-enkephalin was short lasting and was observed 10 to 15 min after i.c.v. perfusion. Morphiceptin had a relatively longer duration of antinociceptive action, the effect was observed 20-50 min after i.c.v. perfusion. Neither morphiceptin nor Met-enkephalin produced antinociception after peripheral injections. The results of the present study indicate that both tested peptides act at mu-opioid receptors situated in the central nervous system. They also suggest that mu-opioid receptors present in the central nervous system are an important element of the trigemino-hypoglossal reflex arc. For that reason selective mu-opioid receptor ligands, like morphiceptin, inhibit the reflex more significantly.

Somatostatin analogs.

Somatostatin is a hypothalamic peptide hormone that inhibits the secretion of growth hormone, glucagon, insulin, gastrin and secretin, and also plays a role in neural transmission. Because of its wide range of possible clinical applications hundreds of somatostatin analogs have been synthesized and bioassayed to date. This review gives a historical perspective, summarizing approximately 30 years of research on somatostatin. The main focus is on the structure-activity relationships and conformational studies of the last generation of somatostatin agonists and their selectivity for five somatostatin receptor subtypes. Achievements in the synthesis of nonpeptide somatostatin analogs, as well as the first somatostatin antagonists, are also discussed. Finally, the use of a cyclic somatostatin scaffold to design ligands for other G-protein-coupled receptors, such as opioid and melanocortin receptors, is mentioned.

Synthesis and biological activity of highly potent heptapeptide analogs of somatostatin with C-terminal modifications.

OBJECTIVE: Structure-activity relationship studies of a series of new heptapeptide analogs of somatostatin with C-terminal modifications were performed. METHODS: New somatostatin analogs were synthesized by solid-phase method. The suppression of the release of growth hormone by the new analogs was assayed in vivo in rats, and the duration of action was investigated using growth hormone time-course assay. Growth hormone concentration in plasma samples was determined by RIA. RESULTS: The growth hormone release inhibitory activity of the new analogs was up to 50 times greater than that of somatostatin. Hydrophilic substitutions at the C-terminus seemed to be advantageous for potency. The most potent analog of the series, D-Phe-c[Cys-Phe-D-Trp-Lys-Thr-Cys]-NH-CH2-CH2-OH showed the most prolonged activity by inhibiting the release of growth hormone for at least 3 h. The analogs containing D-Phe or D-Tyr at the N-terminus were almost equipotent, which proves that the exocyclic N-terminal residue is not involved directly in the receptor recognition. CONCLUSIONS: New heptapeptide analogs of somatostatin with C-terminal modifications were more potent and longer acting than the native hormone.

Effect of cerebral ventricles perfusion with naloxone on trigemino-hypoglossal reflex in rats.

The goal of this study was to determine whether opioid receptor antagonist naloxone abolishes the influence of periaqueductal central gray (PAG) on nociceptive evoked tongue jerks (ETJ) -- a trigemino-hypoglossal reflex induced by tooth pulp stimulation. In rats under chloralose anesthesia three series of experiments were performed. In the first two groups perfusions of lateral ventricles-cerebellomedullary cistern with McIlwain-Rodnight's solution and naloxone were carried out. In group 3 naloxone was infused through a catheter through the jugular vein. The amplitudes of tongue jerks induced by tooth pulp stimulation were recorded during subsequent 10 min perfusions. Mean amplitude of tongue movements induced by tooth pulp stimulation was regarded as the indicator of the magnitude of trigemino-hypoglossal reflex. We observed that perfusion of the cerebral ventricles with naloxone (100 nmol/ml) increased the trigemino-hypoglossal reflex up to 143%. The amplitude of ETJ was significantly reduced during PAG stimulation with a train of electrical impulses. After obtaining a significant -- 93% -- inhibition of ETJ (7% of the control), naloxone (100 nmol/ml) was added to the perfusion fluid. This led to a significant increase of the reflex up to 68%. Infusion of naloxone through the jugular vein did not affect the reflex. The above results suggest that the inhibition of ETJ due to PAG stimulation is partially reversed by naloxone and mediated via interactions with endogenous opioid systems involved in modulation of nociception.

The effect of naloxone on trigemino-hypoglossal reflex inhibited by periaqueductal central gray stimulation in rats.

The aim of the study was to determine whether opioid receptor antagonist naloxone abolishes the influence of periaqueductal central gray (PAG) on nociceptive evoked tongue jerks (ETJ) - a trigemino-hypoglossal reflex induced by tooth pulp stimulation. In rats under chloralose anesthesia three subsequent series of perfusions of lateral ventricles - cerebellomedullary cistern with Mc Ilwain-Rodnight's solution, Met-enkaphalin (Enk-Met) and naloxone were carried out. The amplitudes of tongue jerks induced by tooth pulp stimulation were recorded during subsequent 10 min perfusions. Mean amplitude of tongue movements induced by tooth pulp stimulation was regarded as the indicator of the magnitude of trigemino-hypoglossal reflex. We observed that perfusion of the cerebral ventricles with Enk-Met (100 nmol/mL) inhibited the trigemino-hypoglossal reflex by 46%, whereas naloxone (100 nmol/mL), added to the solution perfusing the cerebral ventricles system, increased the reflex by 42%. The amplitude of ETJ was significantly reduced during PAG stimulation with a train of electrical impulses. After obtaining a significant 93% - inhibition of ETJ, naloxone (100 nmol/mL) was added to the perfusion fluid. This led to a significant increase of the reflex by 68%. The above results suggest that the inhibition of ETJ due to PAG stimulation is partially reversed by naloxone and mediated via interactions with endogenous opioid systems involved in modulation of nociception.