The N-type calcium channel is a novel target for treating alcohol use disorders

We recently validated the N-type calcium channel as a target for the treatment of alcoholism and anxiety. N-type calcium channels are neuronal presynaptic ion channels that regulate neurotransmitter release at many sites in the brain. Mice lacking N-type calcium channels exhibit reduced ethanol consumption and show resistance to the acute intoxicating effects of ethanol. In wild type rodents, pretreatment with a novel N- and T-type calcium channel blocker, NP078585, reduces the intoxicating and reinforcing effects of ethanol and abolishes stress-induced reinstatement of alcohol seeking. Here we discuss these findings and expand upon their implications for the N-type calcium channel as a target for drug development. An important consideration in the development of drugs to treat any addiction is that the medication itself not be addictive. We attempted, and failed, to generate a conditioned place preference for NP078585, suggesting that NP078585 is not rewarding.     

Ghrelin as a target for gastrointestinal motility disorders

The therapeutic potential of ghrelin and synthetic ghrelin receptor (GRLN-R) agonists for the treatment of gastrointestinal (GI) motility disorders is based on their ability to stimulate coordinated patterns of propulsive GI motility. This review focuses on the latest findings that support the therapeutic potential of GRLN-R agonists for the treatment of GI motility disorders. The review highlights the preclinical and clinical prokinetic effects of ghrelin and a series of novel ghrelin mimetics to exert prokinetic effects on the GI tract. We build upon a series of excellent reviews to critically discuss the evidence that supports the potential of GRLN-R agonists to normalize GI motility in patients with GI hypomotility disorders such as gastroparesis, post-operative ileus (POI), idiopathic chronic constipation and functional bowel disorders.     

Pendrin as a novel target for diuretic therapy

The Cl(-)/HCO(3)(-) exchanger pendrin (SLC26A4, PDS) and the thiazide-sensitive NaCl cotransporter NCC (SLC12A3) are expressed on the apical membranes of distal nephron segments and mediate salt absorption, with pendrin working in tandem with the epithelial Na channel (ENaC) and NCC working by itself. Pendrin is expressed on the apical membrane of intercalated cells in late distal convoluted tubule (DCT), connecting tubule (CNT) and the cortical collecting duct (CCD) whereas the thiazide-sensitive NaCl cotransporter NCC is primarily detected on the apical membrane of DCT cells. Recent studies indicate that pendrin expression is increased in kidneys of NCC knockout mice, raising the possibility that pendrin and NCC can compensate for loss of the other by increasing their expression and activity. Current investigations in our laboratories demonstrate that pendrin plays an important role in compensatory salt absorption in response to the loop diuretics and the thiazide derivatives. These studies further demonstrate that whereas single deletion of pendrin or NCC does not cause salt wasting in mutant mice under baseline conditions, double knockout of pendrin and NCC causes profound polyuria and polydipsia, along with salt wasting under basal conditions. As a result, animals develop significant dehydration. We propose that pharmacologic inhibition of pendrin and NCC can provide a novel and strong diuretic regimen for patients with fluid overload, including those with congestive heart failure, nephrotic syndrome or renal failure.     

Efficacy of the alcohol use disorders identification test as a screening tool for hazardous alcohol intake and related disorders in primary care: a validity study.

OBJECTIVE: To determine the properties of the alcohol use disorders identification test in screening primary care attenders for alcohol problems. DESIGN: A validity study among consecutive primary care attenders aged 18-65 years. Every third subject completed the alcohol use disorders identification test (a 10 item self report questionnaire on alcohol intake and related problems) and was interviewed by an investigator with the composite international diagnostic interview alcohol use module (a standardised interview for the independent assessment of alcohol intake and related disorders). SETTING: 10 primary care clinics in Verona, north eastern Italy. PATIENTS: 500 subjects were approached and 482 (96.4%) completed evaluation. RESULTS: When the alcohol use disorders identification test was used to detect subjects with alcohol problems the area under the receiver operating characteristic curve was 0.95. The cut off score of 5 was associated with a sensitivity of 0.84, a specificity of 0.90, and a positive predictive value of 0.60. The screening ability of the total score derived from summing the responses to the five items minimising the probability of misclassification between subjects with and without alcohol problems provided an area under the receiver operating characteristic curve of 0.93. A score of 5 or more on the five items was associated with a sensitivity of 0.79, a specificity of 0.95, and a positive predictive value of 0.73. CONCLUSIONS: The alcohol use disorders identification test performs well in detecting subjects with formal alcohol disorders and those with hazardous alcohol intake. Using five of the 10 items on the questionnaire gives reasonable accuracy, and these are recommended as questions of choice to screen patients for alcohol problems.     

Identification of ATP-synthase as a novel intracellular target for microcystin-LR

Microcystins (MCs) are a group of closely related cyclic heptapeptides produced by a variety of common cyanobacteria. These are potent and highly specific hepatotoxins, the toxicity of which is based upon their inhibition of type-1 (PP1) and type-2A (PP2A) protein phosphatases. Apart from protein phosphatases, it is not known whether these phosphatase-inhibiting peptides could bind any other cellular proteins. We wanted to determine whether any possible unknown MC-adducts could explain the apoptotic effects observed at high concentrations of MCs. The question of other possible cellular proteins binding to MCs is also relevant when these compounds are employed for affinity purification of protein phosphatases. In MC-treated cell lysates, antibodies to MC recognized three protein adducts of 35-37 and 55 kD. By immunochemical and proteomics approaches, these proteins were identified as the catalytic subunits of type-1 and type-2A protein phosphatases and the ATP-synthase beta-subunit. The latter target could be associated with the suggested apoptosis-inducing potential of MCs.     

Midkine as a novel target for antibody therapy in osteosarcoma

Osteosarcoma is a malignant tumor with poor prognosis, and lack of accurate prognostic factors is one of the reasons that make this tumor difficult to cure. The heparin-binding growth factor, midkine is involved in generation and progression of many types of tumors. However, the relationship between midkine and osteosarcoma has been unclear. We show here that midkine is overexpressed in osteosarcoma and the level of midkine expression is correlated with prognosis (P<0.05; log-rank test). Treatment with functional antibodies against midkine suppresses growth of osteosarcoma cell lines, 9N2, 3N1, Saos-2, and NOS-1, to 25-65% of untreated controls. Our results suggest that midkine is useful as a prognostic marker, and is a candidate therapeutic target for osetosarcomas.     

Choline transporter as a novel target for molecular imaging of cancer

Abnormalities of choline processing in cancer cells have been used as a basis for imaging of cancer with positron emission tomography and magnetic resonance spectroscopy. In this study, the transport mechanism for choline was investigated in cultured PC-3 prostate cancer cells. Furthermore, tritiated hemicholinium 3 (HC-3), a well-known inhibitor of choline transport, was studied as a prototypic molecular imaging probe in PC-3 cells and 9L glioma-bearing rats. [(3)H]Choline uptake by PC-3 cells was found to have both facilitative and nonfacilitative components. Facilitative transport was characterized by partial sodium dependence and intermediate affinity (K(M) = 9.7 +/- 0.8 microM). HC-3 inhibited choline with a K(I) of 10.5+/- 2.2 microM. Ouabain (1 mM) caused a 94% reduction in choline uptake. At physiologic choline concentration, phosphocholine was the rapid and predominant metabolic fate. The binding of [(3)H]HC-3 to PC-3 cells was rapid and specific (competitively blocked with unlabeled HC-3). Biodistribution of [(3)H]HC-3 in 9L glioma-bearing rats showed the ranking of uptake to be kidney > lung > tumor > liver > skeletal muscle congruent with blood > brain. In comparison with [(14)C]choline, [(3)H]HC-3 showed over twofold higher tumor uptake and favorable uptake ratios of tumor to blood, tumor to muscle, tumor to lung, and tumor to liver. The data demonstrate the quantitative importance of an intermediate-affinity, partially sodium-dependent choline transport system on choline processing in PC-3 cancer cells. The biodistribution properties of [(3)H]HC-3 in tumor-bearing rats encourage the development of molecular imaging probes based on choline transporter binding ligands.     

Surrogate phenotype definition for alcohol use disorders: a genome-wide search for linkage and association

For the identification of susceptibility loci in complex diseases the choice of the target phenotype is very important. We compared results of genome-wide searches for linkage or for association related to three phenotypes for alcohol use disorder. These are a behavioral score BQ, based on a 12-item questionnaire about drinking behavior and the subject's report of drinking-related health problems, and ERP pattern and ERP magnitude, both derived from the eyes closed resting ERP measures to quantify brain activity. Overall, we were able to identify 11 candidate regions for linkage. Only two regions were found to be related to both BQ and one of the ERP phenotypes. The genome-wide search for association using single-nucleotide polymorphisms did not yield interesting leads.     

ATP-sensitive potassium channel: a novel target for protection against UV-induced human skin cell damage

Ultraviolet radiation (UV) induces cell damages leading to skin photoaging and skin cancer. ATP-sensitive potassium (K(ATP)) channel openers (KCOs) have been shown to exert significant myocardial preservation and neuroprotection in vitro and in vivo, and yet the potential role of those KCOs in protection against UV-induced skin cell damage is unknown. We investigated the effects of pinacidil and diazoxide, two classical KCOs, on UV-induced cell death using cultured human keratinocytes (HaCat cells). Here, we demonstrated for the first time that Kir 6.1, Kir 6.2 and SUR2 subunits of K(ATP) channels are functionally expressed in HaCaT cells and both non-selective K(ATP) channel opener pinacidil and mitoK(ATP) (mitochondrial K(ATP)) channel opener diazoxide attenuated UV-induced keratinocytes cell death. The protective effects were abolished by both non-selective K(ATP) channel blocker glibenclamide and selective mitoK(ATP) channel blocker 5-hydroxydecanoate (5-HD). Also, activation of K(ATP) channel with pinacidil or diazoxide resulted in suppressive effects on UV-induced MAPK activation and reactive oxygen species (ROS) production. Unexpectedly, we found that the level of intracellular ROS was slightly elevated in HaCaT cells when treated with pinacidil or diazoxide alone. Furthermore, UV-induced mitochondrial membrane potential loss, cytochrome c release and ultimately apoptotic cell death were also inhibited by preconditioning with pinacidil and diazoxide, and their effects were reversed by glibenclamide and 5-HD. Taken together, we contend that mitoK(ATP) is likely to contribute the protection against UV-induced keratinocytes cell damage. Our findings suggest that K(ATP) openers such as pinacidil and diazoxide may be utilized to prevent from UV-induced skin aging.     

The spliceosome: a novel multi-faceted target for therapy

The spliceosome is a dynamic and flexible ribonucleoprotein enzyme that removes intronic sequences in a regulated manner. Spliceosome action enables one stretch of genomic DNA sequence to yield several mRNAs that encode different proteins. It depends on a flexible mechanism for selecting splice sites, which calls for regulatory sequences (splicing enhancers or silencers) recognized by cognate trans-acting protein factors and constitutive ribonucleoprotein devices to build up the catalytic core. The identification of both types of elements now offers a comprehensive insight into how the spliceosome is adapted to carry out the removal of different introns and suggests novel therapeutic targets to, ultimately, restore a physiological pattern of alternatively spliced variants in a large repertoire of pathologies.     

Membrane rafts as a novel target in cancer therapy

Membrane rafts are distinct plasma membrane microdomains that are enriched in sphingolipids and cholesterol. They organize receptors and their downstream molecules and regulate a number of intracellular signaling pathways. This review presents information on the dependence of several growth factor receptor signaling pathways on membrane rafts. It also discusses the involvement of rafts in the regulation of differentiation, apoptosis and cell migration connected with invasiveness and metastasis. Examples of known synthetic and naturally occurring substances that are known to affect lateral membrane organization in tumor cell growth are discussed as potential or actual therapeutics.