Excessive alcohol use, a major cause of morbidity and mortality, is less well understood than other addictive disorders. Dopamine release in ventral striatum is a common element of drug reward, but alcohol has an unusually complex pharmacology, and humans vary greatly in their alcohol responses. This variation is related to genetic susceptibility for alcoholism, which contributes more than half of alcoholism risk. Here, we report that a functional OPRM1 A118G polymorphism is a major determinant of striatal dopamine responses to alcohol. Social drinkers recruited based on OPRM1 genotype were challenged in separate sessions with alcohol and placebo under pharmacokinetically controlled conditions, and examined for striatal dopamine release using positron emission tomography and [11C]-raclopride displacement. A striatal dopamine response to alcohol was restricted to carriers of the minor 118G allele. To directly establish the causal role of OPRM1 A118G variation, we generated two humanized mouse lines, carrying the respective human sequence variant. Brain microdialysis showed a four-fold greater peak dopamine response to an alcohol challenge in h/mOPRM1-118GG than in h/mOPRM1-118AA mice. OPRM1 A118G variation is a genetic determinant of dopamine responses to alcohol, a mechanism by which it likely modulates alcohol reward.

Paradigm-shifting modalities to more efficiently deliver drugs to cancerous lesions require the following attributes: nanoscale-size, targetability and stability under physiological conditions. Often, these nanoscale drug delivery vehicles are limited due to agglomeration, poor solubility or cytotoxicity. Thus, we have designed a methodology to encapsulate hydrophobic antineoplastic chemotherapeutics within a 20-30 nm diameter, pH-responsive, non-agglomerating, non-toxic calcium phosphate nanoparticle matrix. In the present study, we report on calcium phosphate nanocomposite particles (CPNP) that encapsulate both fluorophores and chemotherapeutics, are colloidally stable in physiological solution for extended time at 37°C and can efficaciously deliver hydrophobic antineoplastic agents, such as ceramide, in several cell model systems.

Calcium current modulation by the muscarinic cholinergic agonist oxotremorine methiodide (oxo-M) was examined in sympathetic neurons from the superior cervical ganglion of the rat. Oxo-M strongly inhibited calcium currents via voltage-dependent (VD) and voltage-independent (VI) pathways. These pathways could be separated with the use of the specific M(1) acetylcholine receptor antagonist M(1)-toxin and with pertussis toxin (PTX) treatment. Expression by nuclear cDNA injection of the regulator of G-protein signaling (RGS2) or a phospholipase Cbeta1 C-terminal construct (PLCbeta-ct) selectively reduced VI oxo-M modulation in PTX-treated and untreated cells. Expression of the Gbetagamma buffers transducin (Galpha(tr)) and a G-protein-coupled-receptor kinase (GRK3) construct (MAS-GRK3) eliminated oxo-M modulation. Activation of the heterologously expressed neurokinin type 1 receptor, a Galpha(q/11)-coupled receptor, resulted in VI calcium current modulation. This modulation was eliminated with coexpression of Galpha(tr) or MAS-GRK3. Cells expressing Gbeta(1)gamma(2) were tonically inhibited via the VD pathway. Application of oxo-M to these cells produced VI modulation and reduced the amount of current inhibited via the VD pathway. Together, these results confirm the requirement for Gbetagamma in VD modulation and implicate Galpha(q)-GTP and Gbetagamma as components in the potentially novel VI pathway.

Abstract-Cyclic nucleotides are known to modify voltage-gated (L-type) Ca 2ϩ channel activity in vascular smooth muscle cells, but the exact mechanism(s) underlying these effects is not well defined. The purpose of the present study was to investigate the modulatory role of the cAMP-and cGMP-dependent protein kinase (PKA and PKG, respectively) pathways in Ca 2ϩ channel function by using both conventional and perforated-patch-clamp techniques in rabbit portal vein myocytes. The membrane-permeable cAMP derivative, 8-bromo cAMP (0.1 to 10 mol/L), significantly increased (14% to 16%) peak Ba 2ϩ currents, whereas higher concentrations (0.05 to 0.1 mmol/L) decreased Ba 2ϩ currents (23% to 31%). In contrast, 8-bromo cGMP inhibited Ba 2ϩ currents at all concentrations tested (0.01 to 1 mmol/L). Basal Ca V oltage-dependent (L-type) Ca 2ϩ channels play a major role in excitation-contraction coupling in vascular smooth muscle cells. L-type Ca 2ϩ channels are known to be modulated by several intracellular second-messenger systems, including both the cAMP/cAMP-dependent protein kinase (PKA) and cGMP/cGMP-dependent protein kinase (PKG) pathways.1 However, for vascular smooth muscle, little information is known regarding the exact mechanism(s) by which these processes take place. Patch-clamp studies in smooth muscle cells have shown that L-type Ca 2ϩ channel activity can be enhanced by either low concentrations of 8-Br cAMP or the catalytic subunit of PKA. 2-4Stimulation of ␤-adrenergic receptors with Iso has also been shown to increase Ca 2ϩ channel currents. 2,3,[5][6][7] On the other hand, 8-Br cGMP or the NO-releasing agents sodium nitroprusside and SNAP have been reported to lead to a decrease of Ca 2ϩ channel activity. 2,8 -10The precise mechanism underlying the effects of both PKA and PKG on L-type Ca 2ϩ channels remains controversial. A previous study from this laboratory showed that a moderate increase in cAMP elicited with 1 mol/L Iso, 1 mol/L FSK, or 0.1 mmol/L 8-Br cAMP increased Ca 2ϩ channel currents. 2On the other hand, higher levels of cAMP elicited with 10 mmol/L Iso, 10 mol/L FSK, 1 mmol/L 8-Br cAMP, or 0.1 mmol/L 8-Br cGMP led to inhibition of Ca 2ϩ channel currents. Experiments that measured the time course of responses to high concentrations of Iso or FSK revealed that Ca 2ϩ channel currents were initially enhanced and subsequently inhibited. It has been suggested that moderate increases in cAMP enhance Ca 2ϩ channel currents through PKA activation, whereas higher levels of cAMP lead to activation of PKG, which then predominates over the PKA effect (ie, cross activation of PKG by cAMP). Similar findings have been recently reported in colonic smooth muscle cells. 3 In smooth muscle cells from the basilar artery, it has been shown that exposure of inside-out patches to the catalytic subunit of PKA increased L-type Ca 2ϩ channel availability. 4 In apparent conflict with these results, Sperelakis and coworkers [11][12][13] have

In this prospective, observational study we explored whether A118G single nucleotide polymorphism in the human mu-opioid receptor (MOR) gene could explain the inter-individual differences in opioid analgesic requirements in patients with acute postoperative pain and chronic pain. The frequency of the wild-type A118 MOR (major) and variant G118 MOR (minor) alleles in the subjects with chronic, noncancer pain (n = 121) and opioid-naïve subjects with acute postoperative pain (n = 101), serving as the control group, were examined. The relationships among the A118G MOR genotype, opioid requirements, and the numerical pain score were analyzed in both groups. The frequency of the minor allele was significantly lower in the subjects with chronic pain when compared with the group with acute postoperative pain (0.079 versus 0.158; P = 0.009 by chi2 test). No statistically significant association was observed between the presence of A118G MOR polymorphism and the average postoperative pain score or the doses of morphine used in the immediate postoperative period. In the high-quartile, opioid utilization, chronic pain patients, the homozygotic carriers of the major allele required significantly higher opioid dose than did the carriers of the minor allele. The results indicate that although the presence of the minor allele does not appear to affect opioid analgesic use in acute postoperative pain, the minor allele is less common in chronic pain patients, especially in those requiring higher doses of opioid analgesics.

Key pointsr Mechanical and metabolic stimuli from contracting muscles evoke reflex increases in blood pressure, heart rate and sympathetic nerve activity. Little is known, however, about the nature of the mechano-gated channels on the thin fibre muscle afferents that contribute to evoke this reflex, termed the exercise pressor reflex.r We determined the effect of GsMTx4, an inhibitor of mechano-gated Piezo channels, on the exercise pressor reflex evoked by intermittent contraction of the triceps surae muscles in decerebrated, unanaesthetized rats.r GsMTx4 reduced the pressor, cardioaccelerator and renal sympathetic nerve responses to intermittent contraction but did not reduce the pressor responses to femoral arterial injection of compounds that stimulate the metabolically-sensitive thin fibre muscle afferents.r Expression levels of Piezo2 channels were greater than Piezo1 channels in rat dorsal root ganglia.r Our findings suggest that mechanically-sensitive Piezo proteins contribute to the generation of the mechanical component of the exercise pressor reflex in rats.Abstract Mechanical and metabolic stimuli within contracting skeletal muscles evoke reflex autonomic and cardiovascular adjustments. In cats and rats, gadolinium has been used to investigate the role played by the mechanical component of this reflex, termed the exercise pressor reflex. Gadolinium, however, has poor selectivity for mechano-gated channels and exerts multiple off-target effects. We tested the hypothesis that GsMTX4, a more selective mechano-gated channel inhibitor than gadolinium and a particularly potent inhibitor of mechano-gated Piezo channels, reduced the exercise pressor reflex in decerebrate rats. Injection of 10 μg of GsMTx4 into the arterial supply of the hindlimb reduced the peak pressor (control: 24 ± 5, GsMTx4: 12 ± 5 mmHg, P < 0.01), cardioaccelerator and renal sympathetic nerve responses to tendon stretch, a purely mechanical stimulus, but had no effect on the pressor responses to intra-arterial injection of α,β-methylene ATP or lactic acid. Moreover, injection of 10 μg of GsMTx4 into the arterial supply of the hindlimb reduced the peak pressor (control: 24 ± 2, GsMTx4: 14 ± 3 mmHg, P < 0.01), cardioaccelerator and renal sympathetic nerve responses to electrically-induced intermittent hindlimb muscle contractions. By contrast, injection of 10 μg of GsMTx4 into the jugular vein had no effect on the pressor, cardioaccelerator, or renal sympathetic nerve responses to contraction. Quantitative RT-PCR and western blot analyses indicated that both Piezo1 and Piezo2 channel isoforms were natively expressed in rat dorsal root ganglia tissue. We conclude that GsMTx4 reduced the exercise pressor reflex in decerebrate rats and that the reduction was attributable, at least in part, to its effect on mechano-gated Piezo channels.

Activation of several G-protein-coupled receptors leads to voltage-dependent (VD) inhibition of N- and P/Q-type Ca(2+) channels via G-protein betagamma subunits (Gbetagamma). The purpose of the present study was to determine the ability of different Gbetagamma combinations to produce VD inhibition of N-type Ca(2+) channels in rat superior cervical ganglion neurons. Various Gbetagamma combinations were heterologously overexpressed by intranuclear microinjection of cDNA and tonic VD Ca(2+) channel inhibition evaluated using the whole-cell voltage-clamp technique. Overexpression of Gbeta1-Gbeta5, in combination with several different Ggamma subunits, resulted in tonic VD Ca(2+) channel inhibition. Robust Ca(2+) channel modulation required coexpression of both Gbeta and Ggamma. Expression of either subunit alone produced minimal effects. To substantiate the apparent lack of Gbetagamma specificity, we examined whether heterologously expressed Gbetagamma displaced native Gbetagamma from heterotrimeric complexes. To this end, mutant Gbeta subunits were constructed that differentially modulated N-type Ca(2+) and G-protein-gated inward rectifier K(+) channels. Results from these studies indicated that significant displacement does not occur, and thus the observed Gbetagamma modulation can be attributed directly to the heterologously expressed Gbetagamma combinations.

Objectives To assess the reversibility of acute kidney injury (AKI)-induced neutrophil dysfunction and to identify involved mechanisms. Design Controlled laboratory experiment and prospective observational clinical study. Setting University laboratory and hospital. Subjects C57BL/6 wild-type mice. Patients Patients with septic shock with or without AKI. Interventions Murine AKI was induced by i.p. injections of folic acid (nephrotoxic AKI) or by i.m. injections of glycerol (rhabdomyolysis-induced AKI). After 24h, we incubated isolated neutrophils for 3h in normal mouse serum or minimum essential medium (MEM) buffer. We further studied the effects of plasma samples from 13 patients with septic shock (with or without severe AKI) on neutrophilic-differentiated NB4 cells (NB4PMN). Measurements and Main Results Experimental AKI significantly inhibited neutrophil migration and intracellular actin polymerization. Plasma levels of resistin, a pro-inflammatory cytokine and uremic toxin, were significantly elevated during both forms of AKI. Incubation in serum or MEM buffer restored normal neutrophil function. Resistin by itself was able to induce AKI-like neutrophil dysfunction in vitro. Plasma resistin was significantly higher in patients with septic shock with AKI compared to patients with septic shock alone. Compared to plasma from patients with septic shock, plasma from patients with septic shock and AKI inhibited NB4PMN migration. Even after 4d of renal replacement therapy (RRT), plasma from patients with septic shock plus AKI still showed elevated resistin levels and inhibited NB4PMN migration. Resistin inhibited NB4PMN migration and intracellular actin polymerization at concentrations seen during AKI, but not at normal physiological concentrations. Conclusions AKI-induced neutrophil dysfunction is reversible in vitro. However, standard RRT does not correct this defect in patients with septic shock and AKI. Resistin is greatly elevated during AKI, even with ongoing RRT, and is sufficient to cause AKI-like neutrophil dysfunction by itself.