New small molecule fluorescent probes for G protein-coupled receptors: valuable tools for drug discovery.

G protein-coupled receptors (GPCRs) are essential signaling proteins and tractable therapeutic targets. To develop new drug candidates, GPCR drug discovery programs require versatile, sensitive pharmacological tools for ligand binding and compound screening. With the availability of new imaging modalities and proximity-based ligand binding technologies, fluorescent ligands offer many advantages and are increasingly being used, yet labeling small molecules remains considerably more challenging relative to peptides. Focusing on recent fluorescent small molecule studies for family A GPCRs, this review addresses some of the key challenges, synthesis approaches and structure-activity relationship considerations, and discusses advantages of using high-resolution GPCR structures to inform conjugation strategies. While no single approach guarantees successful labeling without loss of affinity or selectivity, the choice of fluorophore, linker type and site of attachment have proved to be critical factors that can significantly affect their utility in drug discovery programs, and as discussed, can sometimes lead to very unexpected results.

Hormonal reactivity during martial arts practice among high-risk youths.

Martial arts have become a popular afterschool activity for youths across the globe. Accumulating data suggest that these activities may confer substantial cognitive and psychological benefits, and recent efforts have been made to introduce martial arts training into educational and rehabilitation settings. However, few studies have examined the potential mechanisms that may underlie these benefits. The current study evaluated the reactivity of two hormones, oxytocin (OT) and cortisol (CT), thought to be respectively involved in regulating mammalian social behaviors and responsivity to stress, to a session of intensive martial arts training in samples of at high-risk and low-risk (in regular educational establishments) youths. OT and CT were measured at baseline, during peak training, and following a cool down period. Analyses revealed that high-risk youths had lower OT but similar CT baseline levels, compared to low-risk youths, prior to the martial arts session. A significant group by time interaction indicated that whereas the OT levels among low-risk youths returned to baseline levels following training, OT levels among high-risk youths remained elevated. Finally, unlike low-risk youths for whom CT levels continued to increase throughout the training session, high-risk youths showed no significant CT reactivity. This study suggests that some of the beneficial effects of martial arts may be related to hormonal processes, especially increases in OT levels, and highlights the differing effects that training may have in different populations.

Prosocial effects of an oxytocin metabolite, but not synthetic oxytocin receptor agonists, in a mouse model of autism.

Currently, there are no established pharmaceutical strategies that effectively treat social deficits in autism spectrum disorder (ASD). Oxytocin, a neurohormone that plays a role in multiple types of social behaviors, has been proposed as a possible therapeutic against social impairment and other symptoms in ASD. However, from the standpoint of pharmacotherapy, oxytocin has several liabilities as a standard clinical treatment, including rapid metabolism, low brain penetrance, and activity at the vasopressin (antidiuretic hormone) receptors. The present studies describe findings from a preclinical screening program to evaluate oxytocin receptor (OXTR) agonists and oxytocin metabolites for potential clinical use as more optimal treatments. We first investigated two synthetic oxytocin analogs, TC-OT-39 and carbetocin, using in vitro cell-based assays for pharmacological characterization and behavioral tests in the BALB/cByJ mouse model of ASD-like social deficits. Although both TC-OT-39 and carbetocin selectively activate the OXTR, neither synthetic agonist had prosocial efficacy in the BALB/cByJ model. We next evaluated two oxytocin metabolites: OT(4-9) and OT(5-9). While OT(5-9) failed to affect social deficits, the metabolite OT(4-9) led to significant social preference in the BALB/cByJ model, in a dose-dependent manner. The increased sociability was observed at both 24 h and 12 days following the end of a subchronic regimen with OT(4-9) (2.0 mg/kg). Overall, these results suggest that the prosocial effects of oxytocin could be mediated by downstream activity of oxytocin metabolites, raising the possibility of new pathways to target for drug discovery relevant to ASD.

Development and stability of a heat-stable formulation of carbetocin for the prevention of postpartum haemorrhage for use in low and middle-income countries.

Postpartum haemorrhage is a leading cause of maternal death worldwide. Oxytocin, currently the drug of choice for prevention of PPH, requires constant refrigeration. In pursuit of an alternative medicine, Ferring Pharmaceuticals have developed a heat-stable formulation of carbetocin, an oxytocin analogue. This study aimed to define that formulation, and to investigate its stability under ICH climate zone IV conditions (30°C/75% relative humidity) for at least 3 years and at extreme temperatures, such as 60°C, for shorter periods of time. The development resulted in a heat-stable carbetocin formulation consisting of 0.1 mg/mL carbetocin in sodium succinate buffer, mannitol, and methionine. The optimum pH was determined to be pH 5.45 (5.25-5.65). The generated stability data of this formulation show that ≥95% purity of the peptide was maintained for a minimum of 3 years at 30°C, 6 months at 40°C, 3 months at 50°C and 1 month at 60°C. In addition, the heat-stable carbetocin formulation was not sensitive to freezing or light. The reported highly stable peptide formulation facilitates the distribution in low and middle-income countries, where maintaining cold chain distribution is difficult. Ferring Pharmaceuticals, the World Health Organization, and MSD for Mothers have established a collaboration to develop this heat-stable formulation of carbetocin for the prevention of post-partum hemorrhage in women after vaginal childbirth, with the aim of making the medicine available in the public sector of developing countries that have a high burden of maternal mortality.

A Predictive, Quantitative Model of Spiking Activity and Stimulus-Secretion Coupling in Oxytocin Neurons.

Oxytocin neurons of the rat hypothalamus project to the posterior pituitary, where they secrete their products into the bloodstream. The pattern and quantity of that release depends on the afferent inputs to the neurons, on their intrinsic membrane properties, and on nonlinear interactions between spiking activity and exocytosis: A given number of spikes will trigger more secretion when they arrive close together. Here we present a quantitative computational model of oxytocin neurons that can replicate the results of a wide variety of published experiments. The spiking model mimics electrophysiological data of oxytocin cells responding to cholecystokinin (CCK), a peptide produced in the gut after food intake. The secretion model matches results from in vitro experiments on stimulus-secretion coupling in the posterior pituitary. We mimic the plasma clearance of oxytocin with a two-compartment model, replicating the dynamics observed experimentally after infusion and injection of oxytocin. Combining these models allows us to infer, from measurements of oxytocin in plasma, the spiking activity of the oxytocin neurons that produced that secretion. We have tested these inferences with experimental data on oxytocin secretion and spiking activity in response to intravenous injections of CCK. We show how intrinsic mechanisms of the oxytocin neurons determine this relationship: In particular, we show that the presence of an afterhyperpolarization (AHP) in oxytocin neurons dramatically reduces the variability of their spiking activity and even more markedly reduces the variability of oxytocin secretion. The AHP thus acts as a filter, protecting the final product of oxytocin cells from noisy fluctuations.

Human milk H2O2 content: does it benefit preterm infants?

BackgroundHuman milk has a high content of the antimicrobial compound hydrogen peroxide (H2O2). As opposed to healthy full-term infants, preterm neonates are fed previously expressed and stored maternal milk. These practices may favor H2O2 decomposition, thus limiting its potential benefit to preterm infants. The goal of this study was to evaluate the factors responsible for H2O2 generation and degradation in breastmilk.MethodsHuman donors' and rats' milk, along with rat mammary tissue were evaluated. The role of oxytocin and xanthine oxidase on H2O2 generation, its pH-dependent stability, as well as its degradation via lactoperoxidase and catalase was measured in milk.ResultsBreast tissue xanthine oxidase is responsible for the H2O2 generation and its milk content is dependent on oxytocin stimulation. Stability of the human milk H2O2 content is pH-dependent and greatest in the acidic range. Complete H2O2 degradation occurs when human milk is maintained, longer than 10 min, at room temperature and this process is suppressed by lactoperoxidase and catalase inhibition.ConclusionFresh breastmilk H2O2 content is labile and quickly degrades at room temperature. Further investigation on breastmilk handling techniques to preserve its H2O2 content, when gavage-fed to preterm infants is warranted.

Excitatory GABAergic Action and Increased Vasopressin Synthesis in Hypothalamic Magnocellular Neurosecretory Cells Underlie the High Plasma Level of Vasopressin in Diabetic Rats.

Diabetes mellitus (DM) is associated with increased plasma levels of arginine-vasopressin (AVP), which may aggravate hyperglycemia and nephropathy. However, the mechanisms by which DM may cause the increased AVP levels are not known. Electrophysiological recordings in supraoptic nucleus (SON) slices from streptozotocin (STZ)-induced DM rats and vehicle-treated control rats revealed that γ-aminobutyric acid (GABA) functions generally as an excitatory neurotransmitter in the AVP neurons of STZ rats, whereas it usually evokes inhibitory responses in the cells of control animals. Furthermore, Western blotting analyses of Cl- transporters in the SON tissues indicated that Na+-K+-2Cl- cotransporter isotype 1 (a Cl- importer) was upregulated and K+-Cl- cotransporter isotype 2 (KCC2; a Cl- extruder) was downregulated in STZ rats. Treatment with CLP290 (a KCC2 activator) significantly lowered blood AVP and glucose levels in STZ rats. Last, investigation that used rats expressing an AVP-enhanced green fluorescent protein fusion gene showed that AVP synthesis in AVP neurons was much more intense in STZ rats than in control rats. We conclude that altered Cl- homeostasis that makes GABA excitatory and enhanced AVP synthesis are important changes in AVP neurons that would increase AVP secretion in DM. Our data suggest that Cl- transporters in AVP neurons are potential targets of antidiabetes treatments.

The use of heat-induced hydrolysis in immunohistochemistry on angiotensin II (AT1) receptors enhances the immunoreactivity in paraformaldehyde-fixed brain tissue of normotensive Sprague-Dawley rats.

The research on components of the renin-angiotensin system delivered a broad image of angiotensin II-binding sites. Especially, immunohistochemistry (IHC) provided an exact anatomical localization of the AT(1) receptor in the rat brain. Yet, controversial results between in vitro receptor autoradiography and IHC as well as between immunohistochemical studies using various antisera started a vehement discussion concerning specificity and cross-reactivity of these antisera. In particular the magnocellular subdivision of the paraventricular nucleus (PVN) and the supraoptic nucleus (SON) provided controversial results on the localization of AT(1) receptors. Both areas are known for angiotensin II-induced release of vasopressin (VP) and oxytocin (OXT). To evaluate the significance of the appropriate method of antigen retrieval and its relevance for the detection of AT(1) receptors we performed IHC on AT(1) receptors in paraformaldehyde-fixed and paraffin-embedded brain tissue of Sprague-Dawley rats using either the detergent Triton X-100 or microwave oven heating. This study demonstrates that heat-induced hydrolysis enhances the quality and quantity of immunoreactivity (IR) in IHC on AT(1) receptors. In the organum vasculosum lamina terminalis and in the parvocellular subdivisions of the PVN we report a distribution of AT(1)-like-IR similar to that observed with other methods. However, in addition, we provide evidence that distinct AT(1)-like-IR is also localized in few magnocellular neurons of the PVN and in few parvocellular neurons of the dorsal SON but not in magnocellular neurons of the SON. Moreover, parallel IHC indicates that few magnocellular OXT- or VP-releasing neurons of the PVN as well as parvocellular OXT-releasing neurons of the SON do also contain AT(1) receptors.

Cloning of pro-vasotocin and pro-isotocin cDNAs from the flounder Platichthys flesus; levels of hypothalamic mRNA following acute osmotic challenge.

Sequences coding for pro-vasotocin and pro-isotocin have been identified by screening a flounder (Platichthys flesus) hypothalamic cDNA library. The 1074-bp proVT and 727-bp proIT sequences contain a signal peptide and hormone, connected to a neurophysin by a Gly-Lys-Arg sequence. Both sequences also have an elongated carboxyl-terminal with a leucine-rich core resembling copeptin but lacking the amino terminal Arg residue. The levels of pro-vasotocin and pro-isotocin mRNA in the hypothalamus were measured concomitantly with pituitary AVT content and plasma AVT concentration following acute transfer of fish between freshwater and seawater. Three days after transfer from seawater to freshwater there appears to be a down regulation of the AVT hormone system with a fall in hypothalamic pro-vasotocin mRNA levels, an increase in pituitary AVT content, and a fall in plasma levels, but these changes did not achieve statistical significance compared to controls. No change in the AVT system was detected 3 days following the transfer of fish from freshwater to seawater. Hypothalamic isotocin mRNA levels did not change following hypo- or hyperosmotic challenge.

Effects of annetocin, an oxytocin-related peptide isolated from the earthworm Eisenia foetida, and some putative neurotransmitters on gut motility of the earthworm.

Annetocin, an oxytocin-related peptide recently isolated from the lumbricid earthworm Eisenia foetida, and putative transmitter substances were examined for their effects on rhythmic, spontaneous contractions of isolated gut preparations of the earthworm. Significant, dose-dependent effects of the following substances were observed: acetylcholine (ACh), gamma-aminobutyric acid (GABA), and dopamine were excitatory, while serotonin (5-HT) and octopamine were inhibitory. Annetocin, oxytocin, and vasotocin stimulated spontaneous contraction of the earthworm gut, annetocin being approximately 10-fold more potent than oxytocin or vasotocin. However, arginine-vasopressin (Arg-vasopressin), lysine-vasopressin (Lys-vasopressin), tocinoic acid (N-terminal hexapeptide fragment of oxytocin), and MSH release-inhibiting factor (MIF; C-terminal tripeptide fragment of oxytocin) did not show any effect on the earthworm gut motility. On the other hand, oxytocin, vasotocin, Arg-vasopressin, Lys-vasopressin, and tocinoic acid caused spontaneous contractions of isolated rat uterine preparations, where the potency was in this order, while annetocin and MIF exerted no oxytocic activity on the uterus. Dose-response relationship of the effects of annetocin and its related peptides on the annelid and mammalian systems shows that amino acid residue at the third position of these peptides is important for exertion of excitatory action on the smooth muscle systems. The results in the present study suggest that receptors for annetocin and for GABA on the earthworm gut, unlike those for ACh, desensitize during continuous exposure to these substances.

Oxytocin antagonists in preterm labour and delivery.

Evidence has been gained that an oxytocin receptor antagonist given by intravenous infusion effectively stops uterine contractions in threatened as well as in actual preterm labour. The findings suggest that the increase of oxytocin receptors is aetiologically important in uncomplicated preterm labour. Oxytocin antagonists could therefore be an attractive alternative to currently used drugs by virtue of their high specificity and lack of serious side-effects. Their use in prophylactic and maintenance therapy may be greater when modified analogues have been developed that allow non-parenteral therapy.

Galanin immunoreactive neurons in the human hypothalamus: colocalization with vasopressin-containing neurons.

Galanin (GA) is a recently described neuropeptide that has been demonstrated to be widely distributed in the hypothalamus of experimental animals. So far there is no immunohistochemical description of GA in the human hypothalamus and, in particular, no studies of the colocalization of this neuropeptide with other transmitter candidates in the human hypothalamus. We have now investigated this question immunohistochemically by using human brains fixed by vascular perfusion within 24 hours of death. Nerve cell bodies and fibers stained for GA were observed throughout the hypothalamus. Major populations of GA-ir cell bodies were found in the suprachiasmatic, intermediate, supraoptic, paraventricular, arcuate, tuberomammillary, and supramammillary nuclei. Scattered positive neurons were found in the periventricular preoptic area, the posterior hypothalamic nucleus, the lateral hypothalamic area, and zona incerta. A few positive cells were located in the dorsomedial and ventromedial hypothalamic nuclei. The number of GA-ir neurons estimated from three brains was 11,100 +/- 2,400 for the intermediate nucleus, 57,800 +/- 9,100 for the supraoptic nucleus and 47,400 +/- 13,900 for the paraventricular nucleus. GA-ir fibers were widely distributed in the hypothalamus. They were more dense in the periventricular and medial hypothalamic zones, whereas the lateral tuberal nuclei and the dorsolateral part of the supraoptic nucleus contained sparse positive fibers. The mammillary complex contained almost no GA-ir fibers. In the ventromedial tuberal region, GA-ir axons formed bundles travelling down in the infundibular stem. In the median eminence the vascular plexus was wrapped by GA-ir fiber networks. The coexistence of GA with arginine vasopressin (AVP), oxytocin (OXY), and tyrosine hydroxylase (TH) was examined in the supraoptic, paraventricular, and suprachiasmatic nuclei in adjacent paraffin sections. Neurons containing both GA and AVP were very common in the supraoptic nucleus and also occurred in the paraventricular and suprachiasmatic nuclei. The supraoptic and paraventricular nuclei also contained some neurons immunoreactive for both GA and OXY. Neurons positive for GA and TH were rare. The topographic distribution of GA-ir neuronal structures in the hypothalamus and the colocalization of GA, principally with AVP and to a lesser extent with OXY, in some hypothalamic nuclei constitute anatomical evidence that this neuropeptide may be involved in the regulation of endocrine, autonomic, and behavioural homeostatic responses.