Is There a Seasonal Influence on Orthopaedic Surgical Wound Infection Rates?

Postoperative surgical wound infections are a significant cause of morbidity in orthopaedic surgical cases. To date, there has been no large, single-institution study evaluating orthopaedic surgical wound infection rates and their correlation with seasonality. The hypothesis of this study was that there would be more infections in the warmer months of the year. A retrospective review of all orthopaedic surgery cases at the authors' institution from 1992 to 2012 was performed of all patients with postoperative wound infections. Patients were placed into two groups on the basis of the date of initial surgical fixation: those occurring in warm months (May-September) and those occurring in cold months (October-April). From July 2010 to June 2012, there was not a statistically significant increase in total infection rate during the months of May to September compared with the months of October to April (0.8% and 0.6%, respectively; p = .131). The hypothesis was rejected: there was no significant increase in post-operative infections during the warmer months. Although previous studies have demonstrated such an increase, the results of this study, which were from a much larger cohort, disagree.

Acetabular dysplasia and posterior wall fractures: The missing link?

BACKGROUND: Traumatologists are unable to predict hip instability based on CT scans and standard radiographs in posterior wall (PW) fractures comprising <50-60 % of the wall, necessitating an examination under anesthesia (EUA). Risk factors for instability have not been clarified, but acetabular dysplasia has been theorized as a potential etiology. Unfortunately, dysplasia is difficult to evaluate in the traumatic setting. The purpose of this study was to compare acetabular morphology between unstable and stable fractures with a novel method to detect dysplasia. METHODS: Patients ≥ 18 years old with a PW fracture that underwent an EUA from 2013 to 2023 were retrospectively identified. For our experimental measurements, the axial distances on CT between the acetabular dome, lateral acetabular opening, and femoral head vertex were recorded. Acetabular geometry was quantified at these levels. Conventional dysplasia metrics (e.g., Tonnis angle) were obtained. Variables were compared between stable and unstable fractures. RESULTS: 58 patients met inclusion criteria with 42 stable versus 16 unstable fractures. Unstable fractures had higher distances between the acetabular dome and femoral head vertex (p > 0.05). They had more cranial fracture exit points (p = 0.0015), lower femoral head coverage (p = 0.0102), and lower posterior acetabular sector angles (p = 0.0281). No other differences in acetabular geometry, demographics, injury characteristics, or other markers of dysplasia were identified. CONCLUSIONS: Unstable hips demonstrated a more recessed acetabular dome when compared to stable hips. Posterior acetabular femoral head coverage and cranial fracture exit point may be related to hip instability. A larger sample size is needed to validate these findings.

Can acetabular dysplasia be measured on axial CT? A measurement for trauma surgeons.

BACKGROUND: Acetabular dysplasia has been theorized as a risk factor instability amongst common acetabular fractures, such as posterior wall (PW) fractures. However, common radiographic measurements often cannot be acquired in trauma patients. We evaluated axial computed tomography (CT) scans to identify novel, easily-obtained measurements that correlate with acetabular dysplasia for use in surgical indications and planning. METHODS: Patients with known acetabular dysplasia undergoing elective periacetabular osteotomy were selected. A different group of patients without pelvic ring or acetabular fractures from an institutional trauma registry were selected as a comparison group. Standard indices of dysplasia were collected, such as center-edge angle (CEA). Acetabular geometric measurements were taken at three axial levels: 0 - cranial CT slice at the dome; 1 - cranial CT where the dome is an incomplete circle; 2 - cranial CT with femoral head visible. Distances between levels were also calculated: Levels 0-1 (Dome Height; DH), Levels 1-2 (Head Height; HH), and Dome-Head Difference (DH - HH = DHD). RESULTS: DH, HH, and DHD were all significantly correlated with CEA, Tonnis angle, and Sharp's angle in dysplastic hips. All dysplastic hips had DH ≤ 2.5 mm and HH ≥ 1.25 mm. DHD ≤ 0 mm was most specific (93.6 % sensitive, 77.3 % specific) for predicting dysplasia. CONCLUSION: DH ≤ 2.5 mm, HH ≥ 1.25 mm, and DHD ≤ 0 mm were independently associated with dysplasia on axial CT scans. These measurements may be quickly and easily used by trauma surgeons to assess a trauma-based axial CT scan for acetabular dysplasia.

Activation of UTP-sensitive P2Y2 receptor induces the expression of cholinergic genes in cultured cortical neurons: a signaling cascade triggered by Ca2+ mobilization and extracellular regulated kinase phosphorylation.

ATP functions as an extracellular signaling molecule that is costored and coreleased with neurotransmitters at central and peripheral neuronal synapses. Stimulation by ATP upregulates the expression of synaptic genes in muscle-including the genes for nicotine acetylcholine receptor (α-, δ-, and ε-subunits) and acetylcholinesterase (AChE)-via the P2Y receptor (P2YR), but the trophic response of neurons to the activation of P2YRs is less well understood. We reported that cultured cortical neurons and the developing rat brain expressed different types of P2YRs, and among these the UTP-sensitive P2Y2R was the most abundant. P2Y2R was found to exist in membrane rafts and it colocalized with the postsynaptic protein PSD-95 in cortical neurons. Notably, agonist-dependent stimulation of P2Y2R elevated the neuronal expression of cholinergic genes encoding AChE, PRiMA (an anchor for the globular form AChE), and choline acetyltransferase, and this induction was mediated by a signaling cascade that involved Ca(2+) mobilization and extracellular regulated kinases 1/2 activation. The importance of P2Y2R action was further shown by the receptor's synergistic effect with P2Y1R in enhancing cholinergic gene expression via the robust stimulation of Ca(2+) influx. Taken together our results revealed a developmental function of P2Y2R in promoting synaptic gene expression and demonstrated the influence of costimulation of P2Y1R and P2Y2R in neurons.

State of the Union: Timeliness to Antibiotics in Open Fractures.

OBJECTIVE: In open fractures, early administration of systemic antibiotics has recently been recognized as a universal recommendation, with the current American College of Surgeons Trauma Center Verification recommendation for administration within 1 hour of facility arrival. We sought to quantify the baseline rate of timely antibiotic administration and the various factors associated with delay. METHODS: Data from the National Trauma Data Bank were obtained for all patients treated for open fractures in 2019. 65,552 patients were included. Univariate and multivariate analyses were performed, first for patient, prehospital, and hospital factors compared with rate of antibiotic administration within 1 hour of hospital arrival, then with a multivariate analysis of factors affecting these times. RESULTS: The overall rate of antibiotic administration within 1 hour of arrival was 47.6%. Patient factors associated with lower rates of timely antibiotics include increased age, Medicare status, and a higher number of comorbidities. Associated prehospital factors included non-work-related injuries, fixed-wing air or police transport, and walk-in arrival method. Patients with lower extremity open fractures were more likely to receive antibiotics within 1 hour of arrival than those with upper extremity open fractures. Traumatic amputations had a higher rate of timely administration (67.3%). ACS trauma Level II (52.5%) centers performed better than Level III (48.3%), Level I (45.5%), and Level IV (34.5%) centers. Multivariate analysis confirmed the findings of the univariate analysis. CONCLUSIONS: Despite current clinical standards, rates of adherence to rapid antibiotic administration are low. Certain patient, facility, and environmental factors are associated with delays in antibiotic administration and can be a focus for quality improvement processes. We plan to use these data to evaluate how focus on antibiotic administration as this quality standard changes practice over time. LEVEL OF EVIDENCE: Prognostic Level III. See Instructions for Authors for a complete description of levels of evidence.

Bone cutting efficiency and heat generation using a traditional fluted Burr and a novel fluteless resurfacing tool.

BACKGROUND: Powered instrumentation is often used for bone preparation and/or removal in many orthopaedic procedures but does risk thermogenesis. This study compares biomechanical properties of a fluted burr and a novel fluteless resurfacing tool. METHODS: Twenty cadaveric metatarsals were tested with four predetermined cutting forces to evaluate heat generation and cutting rate for the fluted burr and fluteless resurfacing tool over 40 s or until a depth of 4 mm was reached. Cutting rate was calculated from displacement transducer data. Heat generation was measured by thermocouples placed in the bone adjacent to the burring site. Assuming a body temperature of 37 °C, a 10 °C increase in heat was used as the threshold of inducing osteonecrosis. FINDINGS: At 1.0 N and 1.7 N, the thermal osteonecrosis threshold was reached at comparable times between burrs, while the bone removed by the resurfacing tool was on average five times greater than fluted burr at 1.0 N and over twice as great at 1.7 N. Statistical analysis of these common cutting forces showed the resurfacing tool had significantly higher cutting rates (P < 0.01). As a result, the fluted burr produced higher temperatures for the same amount of bone removal (P < 0.01). INTERPRETATION: In a cadaveric study, the fluteless resurfacing tool demonstrated higher bone cutting rates and lower heat generation for the same amount of bone removed than a traditional fluted burr.

Adverse Events Following Minimally Invasive Achilles Tendon Repair.

BACKGROUND: The rate of wound complications following traditional open Achilles tendon repair is reported at 7.6%. The purpose of this study is to characterize the rate of wound and other early complications following a specific minimally invasive Achilles tendon repair technique, and to identify any factors associated with increased risk. METHODS: The postoperative courses of 99 patients who underwent minimally invasive Achilles tendon repair by 2 surgeons at separate academic medical centers were retrospectively reviewed. Mean follow-up was 8.1 months (range 3.0-24.6 months). Repair technique was similar in all cases with the exception that 71 procedures used a longitudinal incision and a tourniquet, while 28 procedures used a transverse incision and no tourniquet (surgeon preference). The rates of complications were compared between patients with differing baseline and procedural characteristics. RESULTS: Of the 99 patients included in the study, 2 (2.0%) developed wound complications. There was no statistical difference in the rate of wound complications between patients in the longitudinal incision/tourniquet group and patients in the transverse incision/no tourniquet group (2.8% vs 0%; P = 1.000). Four patients (4.0%) developed sural neuropraxia. One patient developed deep venous thrombosis. There were no cases of rerupture. At final follow-up, all 99 patients had intact Thompson tests and well-healed wounds. CONCLUSIONS: The rate of wound complications following minimally invasive Achilles tendon repair is low at 2.0%. Patients should be counseled that although risk for wound complications may be lower with this minimally invasive technique, there are risks for sural neuropraxia and deep suture reaction. LEVELS OF EVIDENCE: Level III, Retrospective study.

The scaffold protein NHERF2 determines the coupling of P2Y1 nucleotide and mGluR5 glutamate receptor to different ion channels in neurons.

Expressed metabotropic group 1 glutamate mGluR5 receptors and nucleotide P2Y1 receptors (P2Y1Rs) show promiscuous ion channel coupling in sympathetic neurons: their stimulation inhibits M-type [Kv7, K(M)] potassium currents and N-type (Ca(V)2.2) calcium currents (Kammermeier and Ikeda, 1999; Brown et al., 2000). These effects are mediated by G(q) and G(i/o) G-proteins, respectively. Via their C-terminal tetrapeptide, these receptors also bind to the PDZ domain of the scaffold protein NHERF2, which enhances their coupling to G(q)-mediated Ca(2+) signaling (Fam et al., 2005; Paquet et al., 2006b). We investigated whether NHERF2 could modulate coupling to neuronal ion channels. We find that coexpression of NHERF2 in sympathetic neurons (by intranuclear cDNA injections) does not affect the extent of M-type potassium current inhibition produced by either receptor but strongly reduced Ca(V)2.2 inhibition by both P2Y1R and mGluR5 activation. NHERF2 expression had no significant effect on Ca(V)2.2 inhibition by norepinephrine (via alpha(2)-adrenoceptors, which do not bind NHERF2), nor on Ca(V)2.2 inhibition produced by an expressed P2Y1R lacking the NHERF2-binding DTSL motif. Thus, NHERF2 selectively restricts downstream coupling of mGluR5 and P2Y1Rs in neurons to G(q)-mediated responses such as M-current inhibition. Differential distribution of NHERF2 in neurons may therefore determine coupling of mGluR5 receptors and P2Y1 receptors to calcium channels.

ATP induces synaptic gene expressions in cortical neurons: transduction and transcription control via P2Y1 receptors.

Studies in vertebrate neuromuscular synapses have revealed previously that ATP, via P2Y receptors, plays a critical role in regulating postsynaptic gene expressions. An equivalent regulatory role of ATP and its P2Y receptors would not necessarily be expected for the very different situation of the brain synapses, but we provide evidence here for a brain version of that role. In cultured cortical neurons, the expression of P2Y(1) receptors increased sharply during neuronal differentiation. Those receptors were found mainly colocalized with the postsynaptic scaffold postsynaptic density protein 95 (PSD-95). This arises through a direct interaction of a PDZ domain of PSD-95 with the C-terminal PDZ-binding motif, D-T-S-L of the P2Y(1) receptor, confirmed by the full suppression of the colocalization upon mutation of two amino acids therein. This interaction is effective in recruiting PSD-95 to the membrane. Specific activation of P2Y(1) (G-protein-coupled) receptors induced the elevation of intracellular Ca(2+) and activation of a mitogen-activated protein kinase/Raf-1 signaling cascade. This led to distinct up-regulation of the genes encoding acetylcholinesterase (AChE(T) variant), choline acetyltransferase, and the N-methyl-d-aspartate receptor subunit NR2A. This was confirmed, in the example of AChE, to arise from P2Y(1)-dependent stimulation of a human ACHE gene promoter. That involved activation of the transcription factor Elk-1; mutagenesis of the ACHE promoter revealed that Elk-1 binding at its specific responsive elements in that promoter was induced by P2Y(1) receptor activation. The combined findings reveal that ATP, via its P2Y(1) receptor, can act trophically in brain neurons to regulate the gene expression of direct effectors of synaptic transmission.

Associations between upper extremity injury patterns in side impact motor vehicle collisions with occupant and crash characteristics.

INTRODUCTION: Side impact motor vehicle collisions (MVC) represent a significant burden of mortality and morbidity caused by automotive injury within the United States. The objective of this study was to evaluate the relationship between upper extremity (UE) injury patterns and contact sources in side impact MVC with occupant and crash variables. METHODS: Crash Injury Research and Engineering Network data obtained from 1998 to 2012 were used to evaluate UE injuries in side impact crashes. First row drivers and passengers that were at least 16 years old with complete crash information were included. Side impact crashes were defined to have an area of deformation to the side of the vehicle and a principal direction of force between 60° and 120° or 240° and 300°. Injuries were stratified by type, anatomic location, and Abbreviated Injury Scale (AIS) severity. Occupant variables included age, sex, height, weight, body mass index, and Injury Severity Score. Vehicle and crash variables included in the analysis were change in vehicle velocity at the time of impact, maximum door intrusion, maximum B-pillar intrusion, seat track position, belt use, vehicle type, impact type, and injury source. Statistical analysis of the UE injury data included descriptive statistics, linear regression analyses with occupant variables, and logistic regression analyses with vehicle and crash variables. RESULTS: There were 903 UE injuries among 408 case occupants. The most common injury type was soft tissue injury (72.5%). The majority of fractures were proximal to and including the humerus (70.3%) with the clavicle being the most common fracture location (N = 89). AIS 2+ UE injuries were associated with a significantly higher mean occupant Injury Severity Score than AIS 1 UE injuries (p = 0.01). Contact with the door was the leading cause of UE injury (34.2%). The odds (OR [95% confidence interval], p-value) of an AIS 2+ UE injury due to contact with the B-pillar (5.3 [3.1, 9.1], <0.0001), door (1.9 [1.3, 2.7], 0.0006), and steering wheel/assembly (2.7 [1.1, 6.3], 0.03) were significantly higher than all other injury sources combined. Scapula fractures were significantly associated with rearward seat track positions (1.46 [1.04, 2.05], 0.03). CONCLUSIONS: This study provides insight into UE injury patterns in side impact MVC. The clavicle was the most common UE fracture location. Contact with the door resulted in the highest number of UE injuries and the B-pillar resulted in the most severe injuries. Additionally, exposure to greater B-pillar intrusion was associated with increased odds of scapula and clavicle fractures in side impacts.

Activation of P2Y1 nucleotide receptors induces inhibition of the M-type K+ current in rat hippocampal pyramidal neurons.

We have shown previously that stimulation of heterologously expressed P2Y1 nucleotide receptors inhibits M-type K+ currents in sympathetic neurons. We now report that activation of endogenous P2Y1 receptors induces inhibition of the M-current in rat CA1/CA3 hippocampal pyramidal cells in primary neuron cultures. The P2Y1 agonist adenosine 5'-[beta-thio]diphosphate trilithium salt (ADPbetaS) inhibited M-current by up to 52% with an IC50 of 84 nM. The hydrolyzable agonist ADP (10 microM) produced 32% inhibition, whereas the metabotropic glutamate receptor 1/5 agonist DHPG [(S)-3,5-dihydroxyphenylglycine] (10 microM) inhibited M-current by 44%. The M-channel blocker XE991 [10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride] produced 73% inhibition at 3 microM; neither ADPbetaS nor ADP produced additional inhibition in the presence of XE991. The effect of ADPbetaS was prevented by a specific P2Y1 antagonist, MRS 2179 (2'-deoxy-N'-methyladenosine-3',5'-bisphosphate tetra-ammonium salt) (30 microM). Inhibition of the M-current by ADPbetaS was accompanied by increased neuronal firing in response to injected current pulses. The neurons responding to ADPbetaS were judged to be pyramidal cells on the basis of (1) morphology, (2) firing characteristics, and (3) their distinctive staining for the pyramidal cell marker neurogranin. Strong immunostaining for P2Y1 receptors was shown in most cells in these cultures: 74% of the cells were positive for both P2Y1 and neurogranin, whereas 16% were only P2Y1 positive. These results show the presence of functional M-current-inhibitory P2Y1 receptors on hippocampal pyramidal neurons, as predicted from their effects when expressed in sympathetic neurons. However, the mechanism of inhibition in the two cell types seems to differ because, unlike nucleotide-mediated M-current inhibition in sympathetic neurons, that in hippocampal neurons did not appear to result from raised intracellular calcium.

Increased susceptibility to ATP via alteration of P2X receptor function in dystrophic mdx mouse muscle cells.

Pathological cellular hallmarks of Duchenne muscular dystrophy (DMD) include, among others, abnormal calcium homeostasis. Changes in the expression of specific receptors for extracellular ATP in dystrophic muscle have been recently documented: here, we demonstrate that at the earliest, myoblast stage of developing dystrophic muscle a purinergic dystrophic phenotype arises. In myoblasts of a dystrophin-negative muscle cell line established from the mdx mouse model of DMD but not in normal myoblasts, exposure to extracellular ATP triggered a strong increase in cytoplasmic Ca2+ concentrations. Influx of extracellular Ca2+ was stimulated by ATP and BzATP and inhibited by zinc, Coomassie Brilliant Blue-G, and KN-62, demonstrating activation of P2X7 receptors. Significant expression of P2X4 and P2X7 proteins was immunodetected in dystrophic myoblasts. Therefore, full-length dystrophin appears, surprisingly, to play an important role in myoblasts in controlling responses to ATP. Our results suggest that altered function of P2X receptors may be an important contributor to pathogenic Ca2+ entry in dystrophic mouse muscle and may have implications for the pathogenesis of muscular dystrophies. Treatments aiming at inhibition of specific ATP receptors could be of a potential therapeutic benefit.

ATP acts via P2Y1 receptors to stimulate acetylcholinesterase and acetylcholine receptor expression: transduction and transcription control.

At the vertebrate neuromuscular junction ATP is known to stabilize acetylcholine in the synaptic vesicles and to be co-released with it. We have shown previously that a nucleotide receptor, the P2Y1 receptor, is localized at the junction, and we propose that this mediates a trophic role for synaptic ATP there. Evidence in support of this and on its mechanism is given here. With the use of chick or mouse myotubes expressing promoter-reporter constructs from genes of acetylcholinesterase (AChE) or of the acetylcholine receptor subunits, P2Y1 receptor agonists were shown to stimulate the transcription of each of those genes. The pathway to activation of the AChE gene was shown to involve protein kinase C and intracellular Ca 2+ release. Application of dominant-negative or constitutively active mutants, or inhibitors of specific kinases, showed that it further proceeds via some of the known intermediates of extracellular signal-regulated kinase phosphorylation. In both chick and mouse myotubes this culminates in activation of the transcription factor Elk-1, confirmed by gel mobility shift assays and by the nuclear accumulation of phosphorylated Elk-1. All of the aforementioned activations by agonist were amplified when the content of P2Y1 receptors was boosted by transfection, and the activations were blocked by a P2Y1-selective antagonist. Two Elk-1 binding site sequences present in the AChE gene promoter were jointly sufficient to drive ATP-induced reporter gene transcription. Thus ATP regulates postsynaptic gene expression via a pathway to a selective transcription factor activation.

Characterization of the UDP-glucose receptor (re-named here the P2Y14 receptor) adds diversity to the P2Y receptor family.

The cloning of a human G-protein-coupled receptor (GPCR) that specifically responds to UDP-glucose and related sugar-nucleotides has been reported recently. This receptor has important structural similarities to known members of the P2Y receptor family but also shows a distinctly different pharmacological response profile. Here, the IUPHAR Subcommittee for P2Y receptor nomenclature and classification review the current knowledge of this receptor and present their reasons for including this receptor in the P2Y receptor family as the P2Y(14) receptor.

Localization and Quantitative Autoradiography of Glutamatergic Ligand Binding Sites in Chick Brain.

The anatomical localization of glutamate receptor subtype-selective ligand binding sites was investigated in 1-day-old chick brain using quantitative autoradiography. Under the conditions used, the regional distributions of [3H]glutamate, [3H]AMPA (a selective quisqualate receptor ligand) and [3H]kainate binding sites are manifestly different. [3H]l-glutamate binding is densely localized in the telencephalon, particularly in the neostriatum (2.8 pmol/mg protein). In addition, [3H]l-glutamate labels the thalamus, the nucleus mesencephalicus lateralis pars dorsalis, the superficial layers of the optic tectum and the molecular layer of the cerebellum. [3H]AMPA binding sites are most densely localized in the hippocampus (0.90 pmol/mg protein), with an otherwise relatively uniform distribution of binding within the telencephalon. [3H]AMPA also labels the striatum griseum et fibrosum superficiale of the optic tectum and the molecular layer of the cerebellum. [3H]Kainate binding sites are extremely densely packed in the molecular layer of the cerebellum (10 pmol/mg protein). Other regions of [3H]kainate binding include the hyperstriatum and the thalamus. The binding of the NMDA receptor channel blocker [3H]MK-801 is increased in the presence of 1 mM l-glutamate. [3H]MK-801 binding is generally widespread in the telencephalon but is notably absent from the ectostriatum. No evidence of [3H]MK-801 binding sites was detected in the cerebellum, even in the presence of 1 mM l-glutamate. The relatively high densities and the well-defined localizations of the glutamate receptor subtype binding sites suggest that chick brain provides a useful system for the further study of excitatory amino acid receptors.

Mutation of the GABAA receptor M1 transmembrane proline increases GABA affinity and reduces barbiturate enhancement.

All GABA(A) receptor (GABAR) subunits include an invariant proline in a consensus motif in the first transmembrane segment (M1). In receptors containing bovine alpha1, beta1 and gamma2 subunits, we analyzed the effect of mutating this M1 proline to alanine in the alpha1 or beta1 subunit using 3 different expression systems. The beta1 subunit mutant, beta1(P228A), reduced the EC(50) for GABA about 10-fold in whole cell recordings in HEK293 cells and L929 fibroblasts. The corresponding alpha1 subunit mutant (alpha1(P233A)) also reduced the GABA EC(50) when expressed in Xenopus oocytes; alpha1(P233A)beta1gamma2S receptors failed to assemble in HEK293 cells. Binding of [(3)H]flumazenil and [(3)H]muscimol to transfected HEK293 cell membranes showed similar levels of receptor expression with GABARs containing beta1 or beta1(P228A) subunits and no change in the affinity for [(3)H]flumazenil; however, the affinity for [(3)H]muscimol was increased 6-fold in GABARs containing beta1(P228A) subunits. In L929 cells, presence of the beta1(P228A) subunit reduced enhancement by barbiturates without affecting enhancement by diazepam or alfaxalone. Single channel recordings from alpha1beta1gamma2S and alpha1beta1(P228A)gamma2L GABARs showed similar channel kinetics, but beta-mutant containing receptors opened at lower GABA concentrations. We conclude that the beta1 subunit M1 segment proline affects the linkage between GABA binding and channel gating and is critical for barbiturate enhancement. Mutation of the M1 proline in the alpha1 subunit also inhibited receptor assembly.

Coupling of the nucleotide P2Y4 receptor to neuronal ion channels.

1. G protein-linked P2Y nucleotide receptors are known commonly to stimulate the phosphoinositide signalling pathway. However, we have previously demonstrated that the cloned P2Y(2), P2Y(6) and P2Y(1) receptors couple to neuronal N-type Ca(2+) channels and to M-type K(+) channels. Here we investigate the coupling of recombinant, neuronally expressed rat- and human P2Y(4) receptors (rP2Y(4), hP2Y(4)) to those channels. 2. Rat sympathetic neurones were nuclear-injected with a P2Y(4) cDNA plasmid. A subsequent activation of rP2Y(4) or hP2Y(4) by UTP (100 micro M) in whole-cell (ruptured-patch) mode produced only about 12% inhibition of the N-type Ca(2+) current (I(Ca(N))). Surprisingly, in perforated patch mode, UTP produced much more inhibition of I(Ca(N)) (maximally 51%), with an IC(50) value of 273 nM. This inhibition was voltage-dependent and was blocked by co-expression of the betagamma-binding transducin Galpha-subunit. Pertussis toxin (PTX) pretreatment also suppressed I(Ca(N)) inhibition. 3. UTP inhibited the M-current, recorded in perforated patch mode, by (maximally) 52%, with IC(50) values of 21 nM for rP2Y(4) and 28 nM for hP2Y(4). This inhibition was not affected by PTX pretreatment. 4. With rP2Y(4), ATP inhibited the M-current (IC(50) 524 nM, 26 times weaker than UTP), whereas ATP had no agonist activity at hP2Y(4). This suggests a difference in agonist binding site between rP2Y(4) and hP2Y(4). 5. We conclude that, in contrast to other nucleotide receptors studied, the P2Y(4) receptor couples much more effectively to M-type K(+) channels than to Ca(2+) channels. Coupling to the Ca(2+) channels involves the betagamma-subunits of G(i/o)-proteins and requires a diffusible intracellular component that is lost in ruptured-patch recording.

Kinetic analysis of [35S]dATP alpha S interaction with P2y(1) nucleotide receptor.

The kinetics of 2'-deoxyadenosine-5'-O-(1-thiotriphosphate) ([(35)S]dATP alpha S) interaction with membrane fragments of transfected astrocytoma 1321N1 cells, expressing human P2Y(1) receptors, and the same wild-type cells, not expressing P2Y receptors were studied. Binding of this radioligand was observed with both types of membranes, but sites showing slow on-rate were found only on the transfected cells. These "slow" binding sites behaved as a kinetically homogeneous population and their interaction with the radioligand was shown to occur in two steps, R+A(K(A))<==>RA(k(i))<==>(k(-i))(RA), including the relatively slow isomerization of the complex RA into (RA). Evidence was obtained to assign the isomerized ("slow") binding sites on the transfected cells as P2Y(1) receptor sites, differentiated from other binding sites of non-receptor origin by kinetic analysis, and characterised by the kinetic parameters K(A)=59 +/- 19 nM, k(i)=(9.0 +/- 0.8)10(-3)s(-1) and k(-i)=(3.9 +/- 0.7)10(-3)s(-1). [(35)S]dATP alpha S binding, with kinetic criteria, can be of value for differentiation of the receptor sites from non-receptor sites and thus provides solid basis for radioligand assay of P2Y(1) receptors.

Analyzing receptor assemblies in the cell membrane using fluorescence anisotropy imaging with TIRF microscopy.

Signaling within and between animal cells is controlled by the many receptor proteins in their membrane. They variously operate as trans-membrane monomers and homo- or hetero-dimers, and may assemble with ion-channels: analyses thereof are needed in studies of receptor actions in tissue physiology and pathology. Interactions between membrane proteins are detectable when pre-labeled with fluorophores, but a much fuller analysis is achievable via advanced optical techniques on living cells. In this context, the measurement of polarization anisotropy in the emitted fluorescence has been the least exploited. Here we demonstrate its methodology and particular advantages in the study of receptor protein assembly. Through excitation in both TIRF and EPI fluorescence illumination modes we are able to quantify and suppress contributions to the signal from extraneous intra-cellular fluorescence, and we show that the loss of fluorescence-polarization measured in membrane proteins reports on receptor protein assembly in real time. Receptor monomers and homo-dimers in the cell membrane can be analyzed quantitatively and for homo-dimers only a single fluorescent marker is needed, thus suppressing ambiguities that arise in alternative assays, which require multiple label moieties and which are thus subject to stoichiometric uncertainty.