Pharmacologic Characterization of Substituted Nitazenes at µ, κ, and Δ Opioid Receptors Suggests High Potential for Toxicity.

The benzimidazole opioids (substituted nitazenes) are highly potent µ opiod receptor (MOR) agonists with heroin- or fentanyl-like effects. These compounds have caused hospitalizations and fatal overdoses. We characterized the in vitro pharmacology and structure-activity relationships of 19 nitazenes with substitutions at three positions of the benzimidazole core. Affinities were assessed using agonist radioligand binding assays at human µ, κ, and Δ opioid receptors (MOR, KOR, and DOR, respectively) heterologously expressed in CHO cells. Notably, for MOR binding, nine substituted nitazenes had significantly higher affinities than fentanyl including N-pyrrolidino etonitazene, N-pyrrilidino isonitazene, and N-desethyl isotonitazene; 13 had subnanomolar affinities. Only metodesnitazene and flunitazene had significantly lower affinities than fentanyl. Affinities for the substituted nitazenes at KOR and DOR relative to MOR were 46- to 2580-fold and 180- to 1280-fold lower, respectively. Functional activities were assessed using [35S]GTPγS binding assays. Four nitazenes had subnanomolar potencies at MOR: N-pyrrolidino etonitazene, N-pyrrilidino isonitazene, N-pyrrilidino protonitazene and N-desethyl isotonitazene. Ten substituted nitazenes had significantly higher potencies than fentanyl. All tested nitazenes were full MOR agonists. Potencies at KOR and DOR relative to MOR were 7.3- to 7920-fold and 24- to 9400-fold lower, respectively. Thus, many of these compounds are high affinity/high potency MOR agonists with elevated potential to elicit toxicity and overdose at low doses. SIGNIFICANCE STATEMENT: Substituted nitazenes are a growing public health threat. Although the 19 nitazenes tested vary in their opioid receptor pharmacology, a number are very high affinity, high potency, and high efficacy compounds- higher than fentanyl. Their pharmacology suggests high potential for harm.

Fentanyl causes naloxone-resistant vocal cord closure: A platform for testing opioid overdose treatments.

BACKGROUND: High doses of the synthetic opioid fentanyl cause rapid and sustained vocal cord closure (VCC) leading to airway obstruction that prevents overdose victims from breathing. This airway effect is not caused by morphine-derived opiates (e.g. heroin), is distinct from respiratory depression, resistant to naloxone, and can be lethal. However, VCC has not been previously included in animal models of opioid overdose. METHODS: Video laryngoscopy was used to monitor vocal cord movement in anesthetized Sprague-Dawley rats. Rats were administered saline, fentanyl (5, 25, or 50 µg/kg) or morphine (5 mg/kg) in an intravenous (IV) bolus delivered over a 10 s period. The mu opioid receptor (MOR) antagonist naloxone was administered as a pre-treatment (1 mg/kg, IV) 5 min prior to fentanyl (25 µg/kg) or a post-treatment (1 and 2 mg/kg) 1 min after fentanyl (25 µg/kg). RESULTS: Fentanyl (25 and 50 µg/kg) caused sustained and lethal VCC within 10 s. Morphine (5 mg/kg) and fentanyl (5 µg/kg) caused only brief laryngospasm with full recovery. Pre-treatment with naloxone (1 mg/kg) prevented fentanyl-induced VCC, but naloxone (1 and 2 mg/kg) was unable to reverse VCC when administered after fentanyl. CONCLUSIONS: These results indicate sustained VCC is a lethal physiological reaction, specific to fentanyl and resistant to naloxone treatment. While pre-treatment with naloxone prevented fentanyl-induced VCC, naloxone was unable to reverse the effect, suggesting a non-opioid receptor-mediated mechanism. These findings demonstrate the necessity of VCC inclusion in animal models of synthetic opioid overdose and the urgent need for more effective treatments for fentanyl-related overdoses.

Fentanyl but not Morphine Interacts with Nonopioid Recombinant Human Neurotransmitter Receptors and Transporters.

Synthetic opioids, including fentanyl and its analogs, have therapeutic efficacy in analgesia and anesthesia. However, their illicit use in the United States has increased and contributed to the number one cause of death for adults 18-50 years old. Fentanyl and the heroin metabolite morphine induce respiratory depression that can be treated with the µ opioid receptor (MOR) antagonist naloxone. With higher or more rapid dosing, fentanyl, more than morphine, causes chest wall rigidity and can also induce rapid onset laryngospasm. Because non-MORs could mediate differing clinical manifestations, we examined the interactions of fentanyl and morphine at recombinant human neurotransmitter transporters, G protein-coupled receptors, and the N-methyl-D-aspartate glutamate receptor. Both drugs were agonists at MOR, κ, and δ opioid receptors. Morphine had little or no affinity at other human receptors and transporters (K i or IC50 value >100 µM). However, fentanyl had K i values of 1407 and 1100 nM at α 1A and α 1B adrenoceptor subtypes, respectively, and K i values of 1049 and 1670 nM at dopamine D4.4 and D1 receptor subtypes, respectively; it also blocked [3H]neurotransmitter uptake by the vesicular monoamine transporter 2 (IC50 = 911 nM). Pharmacokinetic models indicate that these Ki and IC50 values are pharmacologically relevant. Fentanyl had little affinity for other receptors or transporters. Thus, noradrenergic disposition at specific receptor subtypes in relevant organs may play a role in respiratory and cardiothoracic effects of fentanyl. Data suggest that less selective fentanyl receptor pharmacology could play a role in the different clinical effects of morphine compared with fentanyl, including fentanyl-induced deaths after illicit use. SIGNIFICANCE STATEMENT: The synthetic opioid fentanyl induces different clinical effects, including rapid onset muscular rigidity, vocal cord closure, and rapid death, than the heroin metabolite morphine. Our data indicate for the first time that the two drugs have very different effects at recombinant human neurotransmitter receptors and transporters that might explain those clinical differences.

Methamphetamine use alters human plasma extracellular vesicles and their microRNA cargo: An exploratory study.

Methamphetamine (MA) is the largest drug threat across the globe, with health effects including neurotoxicity and cardiovascular disease. Recent studies have begun to link microRNAs (miRNAs) to the processes related to MA use and addiction. Our studies are the first to analyse plasma EVs and their miRNA cargo in humans actively using MA (MA-ACT) and control participants (CTL). In this cohort we also assessed the effects of tobacco use on plasma EVs. We used vesicle flow cytometry to show that the MA-ACT group had an increased abundance of EV tetraspanin markers (CD9, CD63, CD81), but not pro-coagulant, platelet-, and red blood cell-derived EVs. We also found that of the 169 plasma EV miRNAs, eight were of interest in MA-ACT based on multiple statistical criteria. In smokers, we identified 15 miRNAs of interest, two that overlapped with the eight MA-ACT miRNAs. Three of the MA-ACT miRNAs significantly correlated with clinical features of MA use and target prediction with these miRNAs identified pathways implicated in MA use, including cardiovascular disease and neuroinflammation. Together our findings indicate that MA use regulates EVs and their miRNA cargo, and support that further studies are warranted to investigate their mechanistic role in addiction, recovery, and recidivism.

Biochemical and molecular aspects of vascular adrenergic regulation of blood pressure in the elderly.

Hypertension, orthostatic hypotension, arterial insufficiency, and atherosclerosis are common disorders in the elderly that lead to significant morbidity and mortality. One common factor to these conditions is an age-related decline in vascular beta-adrenergic receptor-mediated function and subsequent cAMP generation. Presently, there is no single cellular factor that can explain this age-related decline, and thus, the primary cause of this homeostatic imbalance is yet to be identified. However, the etiology is clearly associated with an age-related change in the ability of beta-adrenergic receptor to respond to agonist at the cellular level in the vasculature. This paper will review what is presently understood regarding the molecular and biochemical basis of age-impaired beta-adrenergic receptor-mediated signaling. A fundamental understanding of why ß-AR-mediated vasorelaxation is impaired with age will provide new insights and innovative strategies for the management of multiple clinical disorders.

2-Hydroxyestradiol slows progression of experimental polycystic kidney disease.

Male gender is a risk factor for progression of polycystic kidney disease (PKD). 17ß-Estradiol (E2) protects experimentally, but clinical use is limited by adverse effects. Novel E2 metabolites provide many benefits of E2 without stimulating the estrogen receptor, and thus may be safer. We hypothesized that E2 metabolites are protective in a model of PKD. Studies were performed in male control Han:SPRD rats, and in cystic males treated with orchiectomy, 2-methoxyestradiol, 2-hydroxyestradiol (2-OHE), or vehicle, from age 3 to 12 wk. Cystic rats exhibited renal functional impairment (∼50% decrease in glomerular filtration and renal plasma flow rates, P < 0.05) and substantial cyst development (20.5 ± 2.0% of cortex area). 2-OHE was the most effective in limiting cysts (6.0 ± 0.7% of cortex area, P < 0.05 vs. vehicle-treated cystic rats) and preserving function, in association with suppression of proliferation, apoptosis, and angiogenesis markers. Downregulation of p21 expression and increased expression of Akt, the mammalian target of rapamycin (mTOR), and some of its downstream effectors were significantly reversed by 2-OHE. Thus, 2-OHE limits disease progression in a cystic rodent model. Mechanisms include reduced renal cell proliferation, apoptosis, and angiogenesis. These effects may be mediated, at least in part, by preservation of p21 and suppression of Akt and mTOR. Estradiol metabolites may represent a novel, safe intervention to slow progression of PKD.

Age-related ß-adrenergic receptor-mediated vasorelaxation is changed by altering G protein receptor kinase 2 expression.

Beta-adrenergic receptor- (ß-AR) mediated vasorelaxation declines with age. This change is likely related to receptor desensitization, rather than down regulation. One kinase responsible for desensitization is G protein receptor kinase 2 (GRK2). We have shown that GRK expression and activity increases with age in Fischer 344 rat aorta. In this study we validated that carotid arteries have similar age-related changes in the ß-AR signaling axis as aorta. This finding allowed use of in vivo infection and delivery of two adenovirus vectors to carotid arteries of 2-month-old (2M) and 12-month-old (12M) male Fischer 344 rats. Adeno-GRK2 was used to overexpress GRK2, and adeno-ß-ARK-ct was used to inhibit GRK2 function. Following a five-day infection, vessels were collected and ex vivo tissue bath was used to evaluate vasoreactivity. We used KCl contracted segments, and determined that overexpression of GRK2 significantly impaired isoproterenol (ISO)-mediated vasorelaxation in both age groups. Maximum relaxation (MAX) to ISO in vessels from 2M decreased from 44% to 21%. MAX to ISO in vessels from 12M decreased from 12% to 6%. Sensitivity (ED50) in vessels from 2M and 12M was also impaired 57%, and 30% respectively. We also determined that expression of adeno-ß-ARK-ct significantly improved ISO-mediated vasorelaxation in both age groups. MAX in vessels from 2M increased from 44% to 58%. MAX in vessels from 12M increased from 15% to 69%. ED50 in vessels from 2M and 12M was also improved 46%, and 50% respectively. These findings further implicate age-related increases in GRK2 expression as an important regulator of the age-related decline in ß-AR-mediated vasorelaxation.

Characterization of clonal vascular smooth muscle cell lines derived from young and old Fischer 344 rats.

A significant finding with aging humans (and aging animal models) is that blood vessels lose their ability to respond to beta-adrenergic receptor stimuli. Therefore, they produce less cyclic adenosine monophosphate (cAMP) and have decreased vasorelaxation with advancing age. This change likely contributes to hypertension, insufficient blood flow, and atherosclerosis. Our goal was to develop a vascular smooth muscle cell culture model that replicates the molecular and biochemical changes observed in blood vessels with advancing age. A clonal selection strategy was used to produce cell lines from 2-, 6-, 12-, and 24-month-old male Fischer 344 rat aortae. Cultures were validated as smooth muscle cells with immunocytochemistry positive for α-actin and negative for von Willebrand factor VIII. Positive staining for G protein-coupled receptor kinase 2 indicated presence of this adrenergic receptor regulator. A total of n = 5 clones from n = 7 animals for each age group were initially analyzed for cAMP accumulation under three conditions: basal, isoproterenol stimulated, and forskolin stimulated. Results found that at passage 3, there was a significant reduction in cAMP accumulation to isoproterenol. However, this reduction disappeared by passage 6. Secondary analysis segregated clones into phenotypic age groups independent of donor animal age. Segregation identified n = 3 clones per group. At passage 3, the age-related change in the beta-adrenergic change was magnified. However, even with segregation, the adrenergic response was lost by passage 6. Our results show that early passaged clonal vascular smooth muscle cell cultures maintain their aging, adrenergic phenotype. Two separate strategies to identify age-representative phenotypes into later passage were unsuccessful.

p21 is decreased in polycystic kidney disease and leads to increased epithelial cell cycle progression: roscovitine augments p21 levels.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disease with few treatment options other than renal replacement therapy. p21, a cyclin kinase inhibitor which has pleiotropic effects on the cell cycle, in many cases acts to suppress cell cycle progression and to prevent apoptosis. Because defects in cell cycle arrest and apoptosis of renal tubular epithelial cells occur in PKD, and in light of earlier reports that polycystin-1 upregulates p21 and that the cyclin-dependent kinase inhibitor roscovitine arrests progression in a mouse model, we asked whether (1) p21 deficiency might underlie ADPKD and (2) the mechanism of the salutary roscovitine effect on PKD involves p21. METHODS: p21 levels in human and animal tissue samples as well as cell lines were examined by immunoblotting and/or immunohistochemisty. Apoptosis was assessed by PARP cleavage. p21 expression was attenuated in a renal tubular epithelial cell line by antisense methods, and proliferation in response to p21 attenuation and to roscovitine was assessed by the MTT assay. RESULTS: We show that p21 is decreased in human as well as a non-transgenic rat model of ADPKD. In addition, hepatocyte growth factor, which induces transition from a cystic to a tubular phenotype, increases p21 levels. Furthermore, attenuation of p21 results in augmentation of cell cycle transit in vitro. Thus, levels of p21 are inversely correlated with renal tubular epithelial cell proliferation. Roscovitine, which has been shown to arrest progression in a murine model of PKD, increases p21 levels and decreases renal tubular epithelial cell proliferation, with no affect on apoptosis. CONCLUSION: The novelty of our study is the demonstration in vivo in humans and rat models of a decrement of p21 in cystic kidneys as compared to non-cystic kidneys. Validation of a potential pathogenetic model of increased cyst formation due to enhanced epithelial proliferation and apoptosis mediated by p21 suggests a mechanism for the salutary effect of roscovitine in ADPKD and supports further investigation of p21 as a target for future therapy.

Effect of age on vascular beta2-adrenergic receptor desensitization is not mediated by the receptor coupling to Galphai proteins.

Beta-adrenergic receptor (beta-AR)-mediated vasorelaxation declines with age. In the vasculature, beta2-AR undergoes protein kinase A-mediated desensitization that causes a switch in the G protein coupled to beta2-AR; Galphai links instead of Galphas. We exposed Fischer 344 rat aortae of increasing age to a desensitizing dose of isoproterenol, and determined its effect on beta2-AR-mediated vasorelaxation. Desensitization decreased beta2-AR-mediated vasorelaxation in young aortae only. Subsequently, we used pertussis toxin to block Galphai to determine whether changes in beta2-AR/G protein coupling occurred. Galphai inhibition did not reverse desensitization or the age-related change, but there appears to be a population of beta2-AR linked to Galphai, as pertussis toxin treatment improved beta2-AR-mediated vasorelaxation in aortae from animals of all ages. These findings suggest aortic beta2-AR in older animals may be maximally desensitized, which would explain impaired vasorelaxation. Our results also imply that protein kinase A-mediated beta2-AR desensitization may not be responsible for the age-related decline.

Altered endothelial nitric oxide synthase targeting and conformation and caveolin-1 expression in the diabetic kidney.

Experimental diabetes is associated with complex changes in renal nitric oxide (NO) bioavailability. We explored the effect of diabetes on renal cortical protein expression of endothelial NO synthase (eNOS) with respect to several determinants of its enzymatic function, such as eNOS expression, membrane localization, phosphorylation, and dimerization, in moderately hyperglycemic streptozotocin-induced diabetic rats compared with nondiabetic control rats and diabetic rats with intensive insulin treatment to achieve near-normal metabolic control. We studied renal cortical expression and localization of caveolin-1 (CAV-1), an endogenous modulator of eNOS function. Despite similar whole-cell eNOS expression in all groups, eNOS monomer and dimer in membrane fractions were reduced in moderately hyperglycemic diabetic rats compared with control rats; the opposite trend was apparent in the cytosol. Stimulatory phosphorylation of eNOS (Ser1177) was also reduced in moderately hyperglycemic diabetic rats. eNOS colocalized and interacted with CAV-1 in endothelial cells throughout the renal vascular tree both in control and moderately hyperglycemic diabetic rats. However, the abundance of membrane-localized CAV-1 was decreased in diabetic kidneys. Intensive insulin treatment reversed the effects of diabetes on each of these parameters. In summary, we observed diabetes-mediated alterations in eNOS and CAV-1 expression that are consistent with the view of decreased bioavailability of renal eNOS-derived NO.

Decline in caveolin-1 expression and scaffolding of G protein receptor kinase-2 with age in Fischer 344 aortic vascular smooth muscle.

Beta-adrenergic receptor (beta-AR)-mediated vasorelaxation declines with age in humans and animal models. This is not caused by changes in expression of beta-AR, G alpha s, adenylyl cyclase, or protein kinase A but is associated with decreased cAMP production. Expression and activity of G protein receptor kinase-2 (GRK-2), which phosphorylates and desensitizes the beta-AR, increases with age in rat aortic tissue. Caveolin scaffolds the beta-AR, GRK, and other proteins within "signaling pockets" and inhibits GRK activity when bound. We questioned the effect of age on caveolin-1 expression and interaction between caveolin-1 and GRK-2 in vascular smooth muscle (VSM) isolated from 2-, 6-, 12-, and 24-mo-old male Fischer 344 rat aorta. Western blot analysis found expression of caveolin-1 declined with age (6-, 12- and 24-mo-old rat aortas express 92, 50, and 42% of 2-mo-old rat aortas, respectively). Results from density-buoyancy analysis showed a lower percentage of GRK in caveolin-1-specific fractions with age (6-, 12- and 24-mo-old rat aortas express 95, 56, and 12% of 2-mo-old rat aortas, respectively). Coimmunoprecipitation confirmed this finding; density of GRK in caveolin-1 immunoprecipitates was 97, 30, and 21% of 2-mo-old aortas compared with 6-, 12- and 24-mo-old animals, respectively. Immunohistocytochemistry and confocal microscopy confirmed that GRK-2 and caveolin-1 colocalize in VSM. These results suggest that in nonoverexpressed, intact tissue, the decline in beta-AR-mediated vasorelaxation may be caused by both a reduction in caveolin-1 expression and a reduction in binding of GRK-2 by caveolin-1. This could lead to an increase in the fraction of free GRK-2, which could phosphorylate and desensitize the beta-AR.

Age-related changes in vascular adrenergic signaling: clinical and mechanistic implications.

A large and growing segment of the general population are age 65 or older, and this percentage will continue to rise. Primary care of this population has, and is becoming a priority for clinicians. Hypertension, orthostatic hypotension, arterial insufficiency, and atherosclerosis are common disorders in the elderly that lead to significant morbidity and mortality. One common factor to these conditions is an age-related decline in beta-adrenergic receptor (beta-AR)-mediated function and subsequent cAMP generation. Presently, there is no single cellular factor that can explain this age-related decline, and thus the primary cause of this homeostatic imbalance is yet to be identified. However, the etiology is clearly associated with an age-related change in the ability of beta-AR receptor to respond to agonist at the cellular level. This article will review what is presently understood regarding the molecular and biochemical basis of age-impaired beta-AR receptor-mediated signaling. A fundamental understanding of why beta-AR-mediated vasorelaxation is impaired with age will provide new insights and innovative strategies for the management of the multiple clinical disorders that effect older people.