The mid-term results of the Carotid Asymptomatic Stenosis (CARAS) observational study.

INTRODUCTION: Carotid endarterectomy (CEA) in patients with asymptomatic carotid stenosis (ACAS) remains a subject of debate. Current recommendations are based on randomized trials conducted over 20 years ago and improvements in medical therapies may have reduced the risk of cerebral ischemic events (CIE). This study presents a mid-term analysis of results from an ongoing prospective observational study of ACAS patients to assess their CIE risk in a real-world setting. METHODS: This is a prospective observational cohort study of patients with ACAS >60 % (NASCET criteria) identified in a single duplex ultrasonography (DUS) vascular laboratory (trial registered: NCT04825080). Patients were not considered for CEA due to their short life expectancy (<3 year) or absence of signs of plaque vulnerability (ulceration, ipoechogenic core). Patient enrollment started in January 2019 and ended in March 2020 with a targeted sample size of 300 patients.A 5-year follow-up was scheduled. Clinical characteristics, risk factors, and medical therapies were documented, and, when necessary, the best medical therapy (BMT), involving antiplatelet agents, blood pressure control, and statins, was recommended during clinical visits. The primary endpoint was to asses CIEs (including strokes, transient ischemic attacks, amaurosis-fugax) ipsilateral to ACAS along with plaque progression rate and patients survival. Follow-up involved annual clinical visit and carotid DUS examination, complemented by telephone interviews at six-month intervals. RESULTS: The study included 307 patients, with an average age of 80 ± 7 years, of whom 55 % were male. Contralateral stenosis exceeding 60 % was present in 61 (20 %) patients. Seventy-seven percent of patients were on BMT. At a mean follow-up of 41±9 months, 7 ispilateral strokes and 9 TIAs occurred, resulting in 14 CIEs (2 patients experienced both TIA and stroke). According to Kaplan-Meier analysis, the 4-year CIE rate was 6±2 %, with an annual CIE rate of 1.5 %. Fifty-eight (19 %) patients had a stenosis progression which was associated with a higher 4-year estimated CIE rate compared to patients with stable plaque (10.3 % vs 3.2 %, P=.01). Similarly, a contralateral carotid stenosis >60 % was associated with a higher 4-year estimated CIE rate: 11.7 % vs 2.9 %, P=.002. These factors were independently associated with high risk for CIE at the multivariate COX analysis: Hazard Ratio (HR): 3.2; 95 % Confidence Interval: 1.1-9.2 and HR: 3.6; 95 % CI: 1.2-10.5. CONCLUSION: The mid-term results of this prospective study suggest that the incidence of CIE in ACAS patients should not be underestimated, with plaque progression and contralateral stenosis serving as primary predictors of CIEs.

Limb salvage and survival after urgent surgical treatment of popliteal artery aneurysm.

BACKGROUND: Popliteal artery aneurysms (PAAs) need urgent treatment in case of acute thrombosis, distal embolization, or rupture. Few data are available in the literature about the treatment results in these scenarios. The aim of the present study was to evaluate an 11-year multicenter experience in the urgent treatment of PAAs. METHODS: All symptomatic PAAs surgically treated in two vascular centers between 2010 and 2021 were retrospectively analyzed. In the postoperative period periodical clinical and Duplex-Ultrasound evaluation were performed. The evaluated endpoint was the outcome of urgent PAAs treatment according to their clinical presentation. Statistical analysis was performed by Kaplan-Meier log-rank evaluation and multivariable Cox regression tests. RESULTS: Sixty-six PAAs needed an urgent repair. Twelve (18%) patients had a PAA rupture and 54 (82%) had an acute limb ischemia (ALI) due to either distal embolization or acute thrombosis. Patients with ALI underwent bypass surgery in 51 (95%) cases, which was associated with preoperative thrombolysis in 18 (31%) cases. A primary major amputation was performed in 3 (5%) cases. The mean follow-up was 52 ± 21 months with an overall 5-year limb salvage of 83 ± 6%. Limb salvage was influenced only by the number of patent tibial arteries (pTA) [5-years limb salvage 0%, 86 ± 10%, 92 ± 8% and 100% in case of 0, 1, 2 or 3 pTA, respectively (P = .001)]. An independent association of number of pTA and limb loss was found [hazard ratio (HR): 0.14 (95% confidence interval (CI) 0.03-0.6), P = .001]. Overall 5-year survival was 71 ± 7%. Ruptured PAAs were associated with lower 5-year survival compared with the ALI group (48 ± 2% vs. 79 ± 7%, P = .001). The number of pTA (33 ± 20%, 65 ± 10%, 84 ± 10% and 80 ± 10% for 0, 1, 2 and 3 pTA, respectively, P = .001) and the thrombolysis (94 ± 6% vs. 62 ± 10%, P = .03) were associated with higher survival in patients with ALI. There was an independent association of number of pTA and long-term survival [HR 0.15 (95% CI 0.03-0.8), P = .03]. CONCLUSIONS: PAA rupture is the cause of urgent PAA treatment in almost one fifth of cases, and it is associated with lower long-term survival. ALI can benefit from thrombolysis, and long-term limb salvage and survival are associated with the number of pTA.

Diagnosis and management of acute conditions of the extracranial carotid artery.

Symptomatic carotid stenosis and carotid dissection are acute conditions of extracranial cerebrovascular vessels determining transient ischemic attack or stroke. Medical, surgical, or endovascular management are different options to treat these pathologies. This narrative review focused on the management, from symptoms to treatment, of the acute conditions of extracranial cerebrovascular vessels, including post-carotid revascularization stroke. Symptomatic carotid stenosis (> 50% according to North American Symptomatic Carotid Endarterectomy Trial criteria) with transient ischemic attack or stroke benefits from carotid revascularization-primarily with carotid endarterectomy associated with medical therapy-within 2 weeks from symptom onset to reduce the risk of stroke recurrence. Different from acute extracranial carotid dissection, medical management with antiplatelet or anticoagulant therapy can prevent new neurologic ischemic events, considering stenting only in case of symptom recurrence. Stroke after carotid revascularization can be associated with the following etiologies: carotid manipulation, plaque fragmentation, or clamping ischemia. Medical or surgical management is therefore influenced by the cause and timing of the neurologic events after carotid revascularization. Acute conditions of the extracranial cerebrovascular vessels include a heterogeneous group of pathologies and correct management can reduce symptom recurrence substantially.

Morphological and Clinical Predictors of Early/Follow-up Failure of the Endovascular Infrarenal Abdominal Aneurysm Repair With Currently Available Endografts.

PURPOSE: To report outcomes of endovascular repair (EVAR) of infrarenal abdominal aortic aneurysms (AAAs) with currently-available endografts and identify predictors of technical/clinical failure. MATERIALS AND METHODS: Patients undergoing EVAR between 2012 and 2020 were prospectively collected and retrospectively analyzed. Technical success (TS: no type I-III endoleaks, renal/hypogastric arteries loss, iliac leg occlusion, conversion to open repair and mortality within 24 postoperative hour), proximal neck-related TS (nr-TS: no proximal type I endoleaks, unplanned renal arteries coverage), and 30-day mortality were assessed as early outcomes. Proximal type I endoleak (ELIa), survival and freedom from reinterventions (FFRs) were assessed during follow-up. Uni/multivariate analysis and Cox-regression were used to identified factors associated with early and follow-up outcomes; FFR and survival were assessed by Kaplan-Meier analysis. RESULTS: A total of 710 were included. Technical success and nr-TS were 692 (98%) and 700 (99%), respectively. The presence of ≥2 hostile anatomical infrarenal neck characteristics was associated with technical failure (odds ratio [OR]: 2.4; 95% confidence interval [CI]: 1.3-4.1; p: 0.007). Infrarenal neck angle >90° (OR: 2.88; 95% CI: 9.6-50.3; p: 0.004), barrel shape (OR: 2.33; 95% CI: 11.1-100.3; p: 0.02) or presence of ≥2 hostile anatomical infrarenal neck characteristics (OR: 2.16; 95% CI: 2.5-5.3; p: 0.03) were independent risk factors for neck-related technical failures. Six (0.8%) patients died within 30 postoperative days. Chronic obstructive pulmonary disease (OR: 16; 95% CI: 1.1-218.3; p: 0.04) and urgent repair (OR: 15; 95% CI: 1.8-119.6; p: 0.01) were independent risk factors for 30-day mortality. The mean follow-up was 53±13 months. There were 12 (1.7%) ELIa during follow-up. Infrarenal neck length <15 mm (hazard ratio [HR]: 2.8; 95% CI: 1.9-9.6; p: 0.005), diameter >28 mm (HR: 2.7; 95% CI: 1.6-9.5; p: 0.006), angle ≥90° (HR: 2.7; 95% CI: 8.3-50.1; p: 0.007), and persistent type II endoleak (HR: 2.9; 95% CI: 1.6-10.1; p: 0.004) were independent risk factors for ELIa. Freedom from reintervention was 91% at 5 years. The ELIa was an independent risk factor for reinterventions during follow-up (HR: 29.5; 95% CI: 1.4-1.6; p<0.001). Survival was 74% at 5 years with 2 cases (0.3%) of late aortic-related mortality. Peripheral arterial occlusive disease (HR: 1.9; 95% CI: 1.4-3.65; p: 0.03), aneurysm diameter ≥65 mm (HR: 2.2; 95% CI: 1.4-3.26; p<0.001), and infrarenal neck length <15 mm (HR: 1.7; 95% CI: 1.2-2.35; p: 0.04) were independent risk factors for mortality during follow-up. CONCLUSION: Endovascular repair with currently-available endografts has high TS and low 30-day mortality. Survival and FFRs were satisfactory at mid-term. Pre/postoperative risk factors for technical and clinical failure were identified and they should be considered in EVAR indication and postoperative management to reduce complications and improve mid-term outcome. CLINICAL IMPACT: Pre and postoperative risk factors for technical and clinical EVAR failure can be identified and they should be considered in EVAR indication and postoperative management to reduce complications and improve mid-term outcome.

Tissue Ki67 proliferative index expression and pathological changes in hemodialysis arteriovenous fistulae: Preliminary single-center results.

BACKGROUND: Arteriovenous fistula (AVF) for hemodialysis integrates outward remodeling with vessel wall thickening in response to drastic hemodynamic changes. Aim of this study is to determine the role of Ki67, a well-established proliferative marker, related to AVF, and its relationship with time-dependent histological morphologic changes. MATERIALS AND METHODS: All patients were enrolled in 1 year and stratified in two groups: (A) pre-dialysis patients submitted to first AVF and (B) patients submitted to revision of AVF. Morphological changes: neo-angiogenesis (NAG), myointimal thickening (MIT), inflammatory infiltrate (IT), and aneurysmatic fistula degeneration (AD). The time of AVF creation was recorded. A biopsy of native vein in Group A and of arterialized vein in Group B was submitted to histological and immunohistochemical (IHC) analysis. IHC for Ki67 was automatically performed in all specimens. Ki67 immunoreactivity was assessed as the mean number of positive cells on several high-power fields, counted in the hot spots. RESULTS: A total of 138 patients were enrolled, 69 (50.0%) Group A and 69 (50.0%) Group B. No NAG or MIT were found in Group A. Seven (10.1%) Group A veins showed a mild MIT. Analyzing the Group B, a moderate-to-severe MIT was present in 35 (50.7%), IT in 19 (27.5%), NAG in 37 (53.6%); AD was present in 10 (14.5%). All AVF of Group B with the exception of one (1.4%) showed a positivity for Ki67, with a mean of 12.31 ± 13.79 positive cells/hot spot (range 0-65). Ki67-immunoreactive cells had a subendothelial localization in 23 (33.3%) cases, a myointimal localization in SMC in 35 (50.7%) cases. The number of positive cells was significantly correlated with subendothelial localization of Ki67 (p = 0.001) and with NA (p = 0.001). CONCLUSIONS: Native veins do not contain cycling cells. In contrast, vascular cell proliferation starts immediately after AVF creation and persists independently of the time the fistula is set up. The amount of proliferating cells is significantly associated with MIT and subendothelial localization of Ki67-immunoreactive cells, thus suggesting a role of Ki-67 index in predicting AVF failure.

Carotid Artery Stenting With Double-Layer Stent: A Systematic Review and Meta-Analysis.

BACKGROUND: Carotid artery stenting (CAS) in the treatment of significant stenosis is a cause of stroke due to both plaque prolapse and cerebral embolization. New types of stents with a double-layer structure have been designed to minimize plaque prolapse and embolization; these double-layer stents (DLSs) should be able to reduce the stroke risk; however, definite data on their performance are scarce in the literature. METHODS: A systematic search was performed through PubMed, Scopus, and Cochrane Library, according to PRISMA guidelines; all studies on CAS with DLS (Roadsaver/Casper or CGuard) up to January 1, 2022, with a cohort of at least 20 patients were considered eligible. The present meta-analysis was approved and registered on PROSPERO register (CRD42022297512). Patients with tandem lesions or complete carotid occlusion were excluded from the study. The 30-day stroke rate after CAS was analyzed evaluating the preoperative symptomatic status and DLS occlusion. The estimated pooled rate of events was calculated by random effect model and moderators were evaluated. RESULTS: A total of 14 studies were included in the meta-analysis for a total of 1955 patients. The estimated overall (95% confidence interval [CI]) stroke rate was 1.4% (0.9%-2.2%, I2 = 0%), which was not influenced by the type of DLS used: CGuard 0.8% (0.4%-1.8%, I2 = 0%) versus Roadsaver/Casper 1.5% (0.7%-3.2%, I2 = 0%), p=0.30. The 30-day estimated stroke rate was 1.5% (0.8%-2.9%, I2 = 0%) in asymptomatic and 1.9% (1.0%-3.6%, I2 = 0%) in symptomatic patients, with no influence by moderators. The 30-day DLS occlusion rate was 0.8% (0.4%-1.8%, I2 = 0%). The publication bias assessment identified asymmetry in the asymptomatic populations. CONCLUSION: The overall 30-day stroke rate in CAS with DLS is low (1.4%), with similar results in symptomatic and asymptomatic patients. Acute occlusion of DLS is rare (0.8%). Further studies are necessary to reduce the publication bias for asymptomatic patients. CLINICAL IMPACT: CAS with DLS is associated to a low rate of 30-day stroke in both symptomatic (1.9%) and asymptomatic (1.5%) patients. The type of DLS (CGuard or Roadsaver/Casper) did not affect the 30-day stroke rate.

Cerebral Ischemic Events Ipsilateral to Carotid Artery Stenosis. The Carotid Asymptomatic Stenosis (CARAS) Observational Study: First Year Preliminary Results.

OBJECTIVE: To report the characteristics of the prospective observational cohort study "Carotid Asymptomatic Stenosis (CARAS)", including patients with asymptomatic carotid stenosis under medical treatment and their first year of follow-up, in order to estimate the risk of cerebral ischemic events. METHODS: This is a prospective observational cohort study of CARAS>60% (Nascet criteria) patients, identified in a single duplex-ultrasonography (DUS) vascular laboratory (trail registration N: NCT04825080). Patient's enrollment started in January 2019 and ended in March 2020 with the follow-up conclusion scheduled in December 2025. The aimed sample size was calculated at 300 patients for a 5-year follow-up. The primary outcome were the incidence of ipsilateral neurologic ischemic events (stokes and transient ischemic attacks [TIA]), plaque progression rate, and survival. The follow-up was scheduled at six-month intervals for clinical visit and annually for DUS examination. RESULTS: a total of 307 patients completed the first follow-up year. The mean age was 81±4 years, 55% were male. Contralateral stenosis >60% was present in 90 (29%) patients. Antiplatelet therapy and statins adherence was 80% and 88%, respectively. During the first year, 3 ispilateral strokes (1%) and 4 TIAs (1.3%) occurred, for a total of 2.3% ipsilateral ischemic events. During the first year, 43 (14%) plaques had a stenosis progression, which was correlated with the occurrence of neurological events (9.3% vs. 1.1%, P=.001, OR: 8.9; 95%CI: 1.9-41); 6 deaths (2%) occurred in the same period. CONCLUSION: the preliminary one-year results of this prospective study suggest that the overall rate of any ipsilateral ischemic event, and specifically ipsilateral strokes, correlates with plaque progression.

Roles of PI3K/AKT/mTOR Axis in Arteriovenous Fistula.

Renal failure is a worldwide disease with a continuously increasing prevalence and involving a rising need for long-term treatment, mainly by haemodialysis. Arteriovenous fistula (AVF) is the favourite type of vascular access for haemodialysis; however, the lasting success of this therapy depends on its maturation, which is directly influenced by many concomitant processes such as vein wall thickening or inflammation. Understanding the molecular mechanisms that drive AVF maturation and failure can highlight new or combinatorial drugs for more personalized therapy. In this review we analysed the relevance of critical enzymes such as PI3K, AKT and mTOR in processes such as wall thickening remodelling, immune system activation and inflammation reduction. We focused on these enzymes due to their involvement in the modulation of numerous cellular activities such as proliferation, differentiation and motility, and their impairment is related to many diseases such as cancer, metabolic syndrome and neurodegenerative disorders. In addition, these enzymes are highly druggable targets, with several inhibitors already being used in patient treatment for cancer and with encouraging results for AVF. Finally, we delineate how these enzymes may be targeted to control specific aspects of AVF in an effort to propose a more specialized therapy with fewer side effects.

Carotid Endarterectomy is often not Possible after an Unheralded Stroke: Unheralded Stroke in Carotid Artery Stenosis.

OBJECTIVES: The ongoing literature recommends carotid endarterectomy (CEA) primarily for patients with neurological symptoms, however CEA can be precluded by the onset of a severe stroke or a total carotid occlusion. The present study aims to evaluate the effect of unheralded strokes in patients with a previously asymptomatic carotid stenosis (ACS) possibly considered for CEA. MATERIALS AND METHODS: From 2009 to 2019, patients with an unheralded stroke from an ACS were considered. By neurological examination, patients were divided in unfit-for-CEA (uCEA) - either for the severity of the stroke (according to modified Rankin-Scale - mRS) or the onset of a total carotid occlusion - and patients submitted to CEA. Predictors for uCEA and stroke severity were evaluated. RESULTS: Over a total of 532 patients with symptomatic carotid stenosis, 277 (52%) with unheralded stroke were included in the study. One hundred and one (36%) were considered uCEA: 64(23%) due to their neurological conditions (mRS:5) and 37 (13%) because of the onset of carotid occlusion. One hundred seventy-six (64%) patients underwent CEA. The preoperative medical therapy was similar in uCEA vs CEA patients. Age≥80 years and female sex were independently associated with uCEA (OR:5.9, 95%CI:3.1-11.4, P<.01; OR:3.9, 95%CI:2.0-7.6, P<.01. respectively). Patients submitted to CEA had mRS: 0-2 in 102(37%) cases and mRS:3-4 in 74 (27%). The contralateral carotid occlusion (CCO) was independently associated with mRS:3-4 (OR:8.4, 95%CI 1.8-79, P=.01). Postoperative stroke rate after CEA was 2.9% (4/167); patients with preoperative mRS:3-4 had a higher risk for postoperative stroke compared to those with mRS:0-2 (5.9% vs. 0%. P=.02). CONCLUSIONS: An unheralded stroke in patients with ACS leads to a severe neurological damage in more than half of cases, either precluding CEA (36%) or increasing the risk of postoperative complications (27%). Female sex, age≥80 and CCO are independent predictors of these occurrences and should be considered in ACS patients.

Dysbindin-1 modifies signaling and cellular localization of recombinant, human D3 and D2 receptors.

Dystrobrevin binding protein-1 (dysbindin-1), a candidate gene for schizophrenia, modulates cognition, synaptic plasticity and frontocortical circuitry and interacts with glutamatergic and dopaminergic transmission. Loss of dysbindin-1 modifies cellular trafficking of dopamine (DA) D2 receptors to increase cell surface expression, but its influence upon signaling has never been characterized. Further, the effects of dysbindin-1 upon closely related D3 receptors remain unexplored. Hence, we examined the impact of dysbindin-1 (isoform A) co-expression on the localization and coupling of human D2L and D3 receptors stably expressed in Chinese hamster ovary or SH-SY5Y cells lacking endogenous dysbindin-1. Dysbindin-1 co-transfection decreased cell surface expression of both D3 and D2L receptors. Further, while their affinity for DA was unchanged, dysbindin-1 reduced the magnitude and potency of DA-induced adenylate cylase recruitment/cAMP production. Dysbindin-1 also blunted the amplitude of DA-induced phosphorylation of ERK1/2 and Akt at both D2L and D3 receptors without, in contrast to cAMP, affecting the potency of DA. Interference with calveolin/clathrin-mediated processes of internalization prevented the modification by dysbindin-1 of ERK1/2 and adenylyl cyclase stimulation at D2L and D3 receptors. Finally, underpinning the specificity of the influence of dysbindin-1 on D2L and D3 receptors, dysbindin-1 did not modify recruitment of adenylyl cyclase by D1 receptors. These observations demonstrate that dysbindin-1 influences cell surface expression of D3 in addition to D2L receptors, and that it modulates activation of their signaling pathways. Accordingly, both a deficiency and an excess of dysbindin-1 may be disruptive for dopaminergic transmission, supporting its link to schizophrenia and other CNS disorders. Dysbindin-1, a candidate gene for schizophrenia, alters D2 receptors cell surface expression. We demonstrate that dysbindin-1 expression also influences cell surface levels of D3 receptors. Further, Dysbindin-1 reduces DA-induced adenylate cylase recruitment/cAMP production and modifies major signaling pathways (Akt and extracellular signal-regulated kinases1/2 (ERK1/2)) of both D2 and D3 receptors. Dysbindin-1 modulates thus D2 and D3 receptor signaling, supporting a link to schizophrenia.

Eyes as gateways for environmental light to the substantia nigra: relevance in Parkinson's disease.

Recent data indicates that prolonged bright light exposure of rats induces production of neuromelanin and reduction of tyrosine hydroxylase positive neurons in the substantia nigra. This effect was the result of direct light reaching the substantia nigra and not due to alteration of circadian rhythms. Here, we measured the spectrum of light reaching the substantia nigra in rats and analysed the pathway that light may take to reach this deep brain structure in humans. Wavelength range and light intensity, emitted from a fluorescent tube, were measured, using a stereotaxically implanted optical fibre in the rat mesencephalon. The hypothetical path of environmental light from the eye to the substantia nigra in humans was investigated by computed tomography and magnetic resonance imaging. Light with wavelengths greater than 600 nm reached the rat substantia nigra, with a peak at 709 nm. Eyes appear to be the gateway for light to the mesencephalon since covering the eyes with aluminum foil reduced light intensity by half. Using computed tomography and magnetic resonance imaging of a human head, we identified the eye and the superior orbital fissure as possible gateways for environmental light to reach the mesencephalon.

Nitric oxide synthase inhibition reverts muscarinic receptor down-regulation induced by pilocarpine- and kainic acid-evoked seizures in rat fronto-parietal cortex.

We investigated how nitric oxide (NO) synthase inhibitor modulates muscarinic receptor expression in epileptic rats. We found that subchronic treatment (4 days) with Nω-nitro-l-arginine reduced the down-regulation of muscarinic receptors induced by pilocarpine and kainic acid in rat fronto-parietal cortex, notwithstanding the dramatic potentiation of seizures induced by both convulsants. Furthermore, functional experiments in fronto-parietal cortex slices, showed that Nω-nitro-l-arginine reduces the down-regulating effect of pilocarpine on carbachol-induced phosphoinositol hydrolysis. Finally, Nω-nitro-l-arginine greatly potentiated the induction of basic fibroblast growth factor (FGF2) by pilocarpine. These data suggest a potential role of NO in a regulatory feedback loop to control muscarinic receptor signal during seizures. The dramatic potentiation of convulsions by NO synthase inhibitors in some animal models of seizures could derive from preventing this feedback loop.

Bright light exposure reduces TH-positive dopamine neurons: implications of light pollution in Parkinson's disease epidemiology.

This study explores the effect of continuous exposure to bright light on neuromelanin formation and dopamine neuron survival in the substantia nigra. Twenty-one days after birth, Sprague-Dawley albino rats were divided into groups and raised under different conditions of light exposure. At the end of the irradiation period, rats were sacrificed and assayed for neuromelanin formation and number of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra. The rats exposed to bright light for 20 days or 90 days showed a relatively greater number of neuromelanin-positive neurons. Surprisingly, TH-positive neurons decreased progressively in the substantia nigra reaching a significant 29% reduction after 90 days of continuous bright light exposure. This decrease was paralleled by a diminution of dopamine and its metabolite in the striatum. Remarkably, in preliminary analysis that accounted for population density, the age and race adjusted Parkinson's disease prevalence significantly correlated with average satellite-observed sky light pollution.

Experimental strategies for studying G protein-coupled receptor homo- and heteromerization with radioligand binding and signal transduction methods.

Before the molecular biology era, functional experiments on isolated organs and radioligand binding and biochemical experiments on animal tissues were widely used to characterize G protein-coupled receptors (GPCRs). The introduction of recombinant cell lines expressing a single GPCR type has been a big step forward for studying both drug-receptor interactions and signal transduction. Before the introduction of the concept of receptor oligomerization, all data generated were attributed to the interaction of drugs with receptor monomers. Now, considerable data must be reinterpreted in light of receptor homo- and heteromerization. In this chapter, we will review some of the methods used to study radioligand binding and signal transduction modifications induced by GPCR homo- and heteromerization.