Potential Sites Of Action Research Articles

247. Expression of components of the hedgehog signalling pathway during murine spermatogenesis

Hedgehog (Hh) signalling is best known for its involvement in regulating patterning, driving cell proliferation, promoting cell survival and directing differentiation during embryonic development.1 The role that Hedgehog signalling plays in the testis is not yet clearly defined, though deletion of one Hedgehog ligand, Dhh, leads to male infertility. The Gli family of zinc finger TFs, consisting of Gli1, Gli2 and Gli3, are mediators of the Hh signalling cascade in vertebrates. We have previously shown that the mRNA transcripts encoding all three Glis in the adult mouse testis are expressed highly in spermatogonia, spermatocytes and to a lower extent in the round spermatids. To understand the potential sites of action of Hh proteins in spermatogenesis, we have extended our analysis to other genes involved in the Hh signalling pathway in the adult mouse testis. Using in situ hybridization, Patched2, a transmembrane receptor for Hh, was detected in spermatogonia and spermatocytes, with an apparently lower expression in the round spermatids. The mRNA of Smoothened, another transmembrane protein which forms a membrane receptor complex with Patched, is highly expressed in spermatogonia and spermatocytes, again showing lower expression in round spermatids and interstitial cells. Fused, a positive regulator of Hh signalling, is highly expressed in spermatogonia and spermatocytes with slightly lower expression in round spermatids. SuFu is a negative regulator of Hh signalling, known to repress Gli1 function in part by tethering it in the cytoplasm. The mRNA encoding SuFu is absent from spermatogonia, detected in spermatocytes and persists in round spermatids where its expression appears highest, suggesting that the SuFu protein may be acting to switch off Hh signalling at that stage of spermatogenesis. Overall, the regulated expression pattern of these genes in the adult mouse testis suggests a role for Hh signalling in the regulation of spermatogenesis. (1)Ruiz i Altaba A. (1999) Trends Genet. 15(10), 418–425.

α7 Nicotinic acetylcholine receptor knockout selectively enhances ethanol-, but not β-amyloid-induced neurotoxicity

The α7 subtype of nicotinic acetylcholine receptor (nAChR) has been implicated as a potential site of action for two neurotoxins, ethanol and the Alzheimer's disease related peptide, β-amyloid. Here, we utilized primary neuronal cultures of cerebral cortex from α7 nAChR null mutant mice to examine the role of this receptor in modulating the neurotoxic properties of subchronic, “binge” ethanol and β-amyloid. Knockout of the α7 nAChR gene selectively enhanced ethanol-induced neurotoxicity in a gene dosage-related fashion. Susceptibility of cultures to β-amyloid induced toxicity, however, was unaffected by α7 nAChR gene null mutation. Further, β-amyloid did not inhibit the binding of the highly α7-selective radioligand, [ 125I]α-bungarotoxin. On the other hand, in studies in Xenopus oocytes ethanol efficaciously inhibited α7 nAChR function. These data suggest that α7 nAChRs modulate the neurotoxic effects of binge ethanol, but not the neurotoxicity produced by β-amyloid. It is hypothesized that inhibition of α7 nAChRs by ethanol provides partial protection against the neurotoxic properties of subchronic ethanol.

Osteolysis—basic science, incidence and diagnosis

Osteolysis is one of the foremost problems limiting the survival of current hip arthroplasty procedures. It is induced by the wear particles and corrosion products which incite an inflammatory response resulting in bone resorption and eventual loosening and failure of the prosthesis. Host factors are presumed to play a role in the process which involves a variety of cytokines and mediators. It is clinically asymptomatic and is diagnosed by serial radiographic follow-up or CT scan. Future research will help to limit the effects of wear particles by identifying the most suitable bearing surfaces. The pathologic cascade of events triggered by wear particles may be a potential site of action for drugs intended to prevent or check the progression of the disease.

Analysis of Drugs and Metabolites in Tissues and Other Solid Matrices

Analysis of solid matrices are typically requested to help understand drug and/or metabolites concentrations at potential sites of action or toxicology, or to perform distribution studies when radiolabel is unavailable or cannot be used. Solid samples pose additional challenges for bioanalysis in terms of preparation of homogeneous QC pools, estimation of extraction recovery, supply of control matrix, and as methods may be complex and time-consuming to perform. A short literature survey is presented together with examples of tissue assays for discovery, GLP and clinical studies.

Peripheral oxyntomodulin reduces food intake and body weight gain in rats.

Oxyntomodulin (OXM) is a circulating gut hormone released post prandially from cells of the gastrointestinal mucosa. Given intracerebroventricularly to rats, it inhibits food intake and promotes weight loss. Here we report that peripheral (ip) administration of OXM dose-dependently inhibited both fast-induced and dark-phase food intake without delaying gastric emptying. Peripheral OXM administration also inhibited fasting plasma ghrelin. In addition, there was a significant increase in c-fos immunoreactivity, a marker of neuronal activation, in the arcuate nucleus (ARC). OXM injected directly into the ARC caused a potent and sustained reduction in refeeding after a fast. The anorectic actions of ip OXM were blocked by prior intra-ARC administration of the glucagon-like peptide-1 (GLP-1) receptor antagonist, exendin(9-39), suggesting that the ARC, lacking a complete blood-brain barrier, could be a potential site of action for circulating OXM. The actions of ip GLP-1, however, were not blocked by prior intra-ARC administration of exendin(9-39), indicating the potential existence of different OXM and GLP-1 pathways. Seven-day ip administration of OXM caused a reduction in the rate of body weight gain and adiposity. Circulating OXM may have a role in the regulation of food intake and body weight.

Effects of stevioside on glucose transport activity in insulin-sensitive and insulin-resistant rat skeletal muscle

Stevioside (SVS), a natural sweetener extracted from Stevia rebaudiana, has been used as an antihyperglycemic agent. However, little is known regarding its potential action on skeletal muscle, the major site of glucose disposal. Therefore, the purpose of the present study was to determine the effect of SVS treatment on skeletal muscle glucose transport activity in both insulin-sensitive lean ( Fa/-) and insulin-resistant obese ( fa/fa) Zucker rats. SVS was administered (500 mg/kg body weight by gavage) 2 hours before an oral glucose tolerance test (OGTT). Whereas the glucose incremental area under the curve (IAUC glucose) was not affected by SVS in lean Zucker rats, the insulin incremental area under the curve (IAUC insulin) and the glucose-insulin index (product of glucose and insulin IAUCs and inversely related to whole-body insulin sensitivity) were decreased ( P< .05) by 42% and 45%, respectively. Interestingly, in the obese Zucker rat, SVS also reduced the IAUC insulin by 44%, and significantly decreased the IAUC glucose (30%) and the glucose-insulin index (57%). Muscle glucose transport was assessed following in vitro SVS treatment. In lean Zucker rats, basal glucose transport in type I soleus and type IIb epitrochlearis muscles was not altered by 0.01 to 0.1 mmol/L SVS. In contrast, 0.1 mmol/L SVS enhanced insulin-stimulated (2 mU/mL) glucose transport in both epitrochlearis (15%) and soleus (48%). At 0.5 mmol/L or higher, the SVS effect was reversed. Similarly, basal glucose transport in soleus and epitrochlearis muscles in obese Zucker rats was not changed by lower doses of SVS (0.01 to 0.1 mmol/L). However, these lower doses of SVS significantly increased insulin-stimulated glucose transport in both obese epitrochlearis and soleus (15% to 20%). In conclusion, acute oral SVS increased whole-body insulin sensitivity, and low concentrations of SVS (0.01 to 0.1 mmol/L) modestly improved in vitro insulin action on skeletal muscle glucose transport in both lean and obese Zucker rats. These results indicate that one potential site of action of SVS is the skeletal muscle glucose transport system.

4-Aminopyridine Transiently Increases Intraocular Pressure in Rabbits

The purpose of this study is to determine the mechanisms of action involved in the ocular hypertension induced by 4-aminopyridine (4-AP), a voltage-dependent potassium (K⁺) channel blocker, in rabbits. Topical application of 4-AP elevated intraocular pressure (IOP). This action caused increases in the aqueous flow rate as well as aqueous levels of protein and norepinephrine. In isolated iris-ciliary body preparations, 4-AP (0.01, 0.1, 1 mmol/l) caused dose-related increases in field-stimulated norepinephrine release by 43, 222 and 243%, respectively. Taken together, the IOP-elevating effect evoked by 4-AP was associated with enhancement of aqueous norepinephrine levels and norepinephrine release from sympathetic nerves of the iris-ciliary body. These results demonstrate that the 4-AP-sensitive K⁺ channels in sympathetic nerves and the ciliary epithelium are the potential sites of action of the 4-AP-induced ocular hypertension.

Cellular Actions of Gabapentin and Related Compounds on Cultured Sensory Neurones

In this review we outline the actions of gabapentin and pregabalin on the excitability of sensory dorsal root ganglion (DRG) neurones in culture and compare these effects with those seen in other neuronal and cultured cell preparations. We also consider the potential mechanisms of action of gabapentin and pregabalin that may contribute to their anti-nociceptive effects. Gabapentin and pregabalin have similar actions and at saturating concentrations do not have additive effects suggesting that they act at the same or closely associated sites. The only high affinity binding sites yet identified for these drugs are α2δ-subunits (types 1 and 2 but not 3 and 4) of voltage-activated Ca2+ channels. Consistent with this are the findings that gabapentin and pregabalin both attenuate Ca2+ influx through voltage-activated channels and these inhibitory actions may in part provide a mechanism by which multiple firing of action potentials in sensory neurones is reduced. However, it is clear that gabapentin and pregabalin have a number of novel characteristics. Although structural analogues of GABA they appear to act independently of GABA receptor activation in sensory neurones. The actions of gabapentin and pregabalin on sensory neurones are not consistent with selective effects on particular high voltage-activated Ca2+ channel subtypes (L, N, P, Q and R) yet a significant proportion (at least 50%) of the whole cell Ca2+ current is insensitive to these drugs. Cell culture conditions can alter Ca2+ channel subunit expression in such a way as to reduce sensitivity to gabapentin and this can be used to identify potential sites of action of this drug. Analysis of subunit mRNA in cell populations with different sensitivities to gabapentin surprisingly indicated that Ca2+ channel b2-subunits as well as α2δ-subunits may determine Ca2+ channel sensitivity to gabapentin in DRG neurones. In addition to Ca2+ channels, gabapentin and pregabalin modulate other whole cell currents, and in particular a delayed enhancement of voltage-dependent K+ conductances has been observed in cultured DRG neurones. The delayed and prolonged features of this enhancement of voltage-activated K+ currents may reflect gabapentin and pregabalin modulating channel activity via intracellular signalling pathways either via a surface receptor interaction leading to the production of a second messenger or by directly acting at intracellular sites. The wide variety of therapeutic effects achieved with gabapentin and pregabalin offer a considerable challenge when trying to determine cellular mechanisms of action of these apparently simple synthetic compounds. Keywords: gabapentin, sensory dorsal root, pregabalin, anti-nociceptive effects

Glutamate release in the nucleus accumbens is involved in behavioral depression during the Porsolt swim test

An abnormality in glutamate function has been implicated in the neural substrate of depressive disorders. To investigate this in rats, the Porsolt swim test was used to assess the role of glutamate in the nucleus accumbens. Glutamate injected into the nucleus accumbens dose-dependently decreased swimming time on the test day (day 2), whereas N-methyl- d-aspartate antagonists dizocilpine and 2-amino-5-phosphonovalerate increased swimming, like an antidepressant. Dizocilpine injected before the conditioning trial (day 1) did not modify the swimming times during the first day but abolished behavioral depression on day 2. Microdialysis coupled to capillary-zone electrophoresis was then used to determine in vivo changes in glutamate release in 1-min samples during the swim test. On day 1, glutamate increased significantly and reached a maximum of 222% after 3 min of swimming. On day 2, baseline glutamate levels were back to normal, but when the animal was placed in the water, glutamate increased to 419% during the first minute, and the animals swam significantly less. For comparison, tail pinch on consecutive days was used as a nonspecific, repeated stressor while accumbens glutamate levels were measured. Tail pinch on the first day increased glutamate similar to the effect obtained during the first day of swimming; however, a second day of tail pinch decreased glutamate levels, instead of the potentiated response observed during the second day of swimming. These results show that accumbens glutamate plays a role in causing the behavioral aspects of depressed behavior as modeled in the swim test. The accumbens may be a potential site of action for drugs that alter behavioral depression.

The Drosophila MicroRNA Mir-14 Suppresses Cell Death and Is Required for Normal Fat Metabolism

MicroRNAs (miRNAs) are small regulatory RNAs that are between 21 and 25 nucleotides in length and repress gene function through interactions with target mRNAs [1, 2]. The genomes of metazoans encode on the order of several hundred miRNAs [3], but the processes they regulate have been defined for only two in C. elegans[4, 5]. We searched for new inhibitors of apoptotic cell death by testing existing collections of P element insertion lines for their ability to enhance a small-eye phenotype associated with eye-specific expression of the Drosophila cell death activator Reaper. Here we report the identification of the Drosophila miRNA mir-14 as a cell death suppressor. Loss of mir-14 enhances Reaper-dependent cell death, whereas ectopic expression suppresses cell death induced by multiple stimuli. Animals lacking mir-14 are viable. However, they are stress sensitive and have a reduced lifespan. Mir-14 mutants have elevated levels of the apoptotic effector caspase Drice, suggesting one potential site of action. Mir-14 also regulates fat metabolism. Deletion of mir-14 results in animals with increased levels of triacylglycerol and diacylglycerol, whereas increases in mir-14 copy number have the converse effect. We discuss possible relationships between these phenotypes.

The chloride conductance channel ClC-K is a specific marker for the Xenopus pronephric distal tubule and duct

Almost nothing is known about the physiology of the pronephros other than that it is efficient at excreting large volumes of dilute urine. The distal segment of pronephric nephrons is the most poorly characterized region of this organ, in part due to the absence of early markers of this structure. In this report the embryonic expression of a Cl − conductance channel, xClC-K, is described with high level transcription of this gene in the distal segment beginning at Xenopus stage 31. xClC-K is also expressed at lower levels in the pronephric duct. This gene serves as a convenient marker for investigating the development of this physiologically critical nephric segment and also serves to identify the potential site of action of a component of the chloride handling system of the pronephros. As ClC-K channels are involved in human kidney disease the frog pronephros may be a useful system in which to study the in vivo activity of this channel.

Allosteric adenosine receptor modulation reduces hypersensitivity following peripheral inflammation by a central mechanism.

Activation of adenosine A1 receptors by endogenous adenosine or synthetic agonists produces antinociception in animal models of acute pain and also reduces hypersensitivity in models of inflammatory and nerve-injury pain. Allosteric adenosine modulators facilitate and potentiate the action of adenosine agonists at the A1 receptors. The purpose of the current study was to examine the effect and site of action for an allosteric adenosine modulator, T62 [2-amino-3-(4-chlorobenzoyl)-5,6, 7,8-tetrahydrobenzothiophene], in rat models of acute pain and inflammation. Intrathecal (i.t.) T62 did not change the withdrawal latency or threshold of normal rats to acute heat or to acute paw pressure. In contrast, i.t. T62 reversed thermal hypersensitivity in carrageenin-inflamed rats. Subcutaneous (s.c.) injection of T62 into the inflamed paw had no such effect. To investigate a potential site of action on nociceptors, single-unit afferent activity to mechanical stimuli on A delta- and C-fibers was examined in normal or carrageenin-inflamed rats before and after intravenous (i.v.) T62 administration. Intravenous T62, 3 mg/kg, had no significant effect in either normal or inflamed conditions. These results support previous studies to suggest that adenosine receptor modulators lack efficacy to acute nociceptive stimuli in the normal condition, but reduce hypersensitivity during inflammation through a central mechanism.

Human urocortin II, a selective agonist for the type 2 corticotropin-releasing factor receptor, decreases feeding and drinking in the rat.

Corticotropin-releasing factor (CRF) has been hypothesized to modulate consummatory behavior through the Type 2 CRF (CRF(2)) receptor. However, behavioral functions subserved by the CRF(2) receptor remain poorly understood. Recently, human urocortin II (hUcn II), a selective CRF(2) receptor agonist, was identified. To study the effects of this neuropeptide on ingestive behavior, we examined the effects of centrally infused hUcn II (i.c.v. 0, 0.01, 0.1, 1.0, 10.0 micro g) on the microstructure of nose-poke responding for food and water in nondeprived, male rats. Malaise-inducing properties of the peptide were monitored using conditioned taste aversion (CTA) testing. To identify potential sites of action, central induction of Fos protein expression was examined. hUcn II dose dependently reduced the quantity and duration of responding for food and water at doses lower (0.01-1.0 micro g) than that forming a CTA (10 micro g). Effects were most evident during hours 4 to 6 of the dark cycle. Meal pattern analysis showed that hUcn II potently (0.1 micro g) increased the satiating value of food. Rats ate and drank smaller and shorter meals without changing meal frequency. Rats also ate more slowly. hUcn II induced Fos in regions involved in visceral sensory processing and autonomic/neuroendocrine regulation and resembling those activated by appetite suppressants. hUcn II is a promising neuropeptide for investigating the role of the CRF(2) receptor in ingestive behavior.

I, 3. The enteric nervous system and infectious diarrhea

Publisher SummaryThis chapter discusses the background knowledge about the enteric nervous system (ENS) as well as the role of ENS in secretory states of the small intestine. The chapter describes the anatomy and physiology of the ENS. A description of the experimental evidence for the involvement of ENS in secretory states of the gut, primarily in cholera toxin-induced secretion that is the most thoroughly investigated secretory state, is presented in the chapter. The chapter focuses on the involvement of ENS in rotavirus (RV) diarrhea. The involvement of the ENS in diarrhea pathophysiology opens up new potential sites of action for drugs in the treatment of intestinal secretory states. The chapter concludes with a discussion of the sites of action for the pharmacological treatment of diarrhea.

Combination Spinal Analgesic Chemotherapy: A Systematic Review

Combination Spinal Analgesic Chemotherapy: A Systematic Review

Combination spinal analgesic chemotherapy: a systematic review.

Combination spinal analgesic chemotherapy: a systematic review.

Cellular apoptosis and organ injury in sepsis: a review.

Cellular apoptosis and organ injury in sepsis: a review.

Preclinical toxicity studies of kahalalide F, a new anticancer agent: single and multiple dosing regimens in the rat.

Kahalalide F (KF) is a new anticancer agent currently in clinical trials for solid tumors, including prostate cancer. During the preclinical development of this drug, the studies reported here were conducted to determine the acute and multiple dose toxicities of KF when administered intravenously (i.v.) to rats. This dosing route is the intended route of clinical administration. KF was administered i.v. to male and female CD rats using single- and multiple-dose (daily for 5 days) schedules. Animals were observed for clinical signs, and body weight, hematology, and clinical chemistry parameters determined. Animals were necropsied, gross observations and organ weights recorded, and numerous tissues were collected and examined microscopically. KF produced lethality at 375 and 450 microg/kg in males and females, respectively, and the maximum tolerated dose (MTD) was estimated to be 300 microg/kg (1800 microg/m(2)). The nervous system appeared to be a potential site of action for the production of lethality. Single-dose administration of KF at 150 and 300 microg/kg produced organ toxicity in which the kidney was the primary target. Injury to distal convoluted tubules was the most toxicologically significant lesion, and was observed on day 4. However, by day 29, resolution of renal toxicity had occurred in the 150-microg/kg group, but only partial resolution was seen at 300 microg/kg. Renal injury correlated with increased serum creatinine, BUN, and kidney weights at 300 microg/kg, indicating impairment of renal function. Subacute, necrotizing inflammation of bone marrow and peritrabecular osteocyte hyperplasia of bone were seen at 300 microg/kg on day 4, with recovery thereafter. Injury to blood vessels and surrounding tissue at the injection site were produced by KF, likely due to local cytotoxicity. In general, reversibility of toxicity was seen at 150 microg/kg but not at 300 microg/kg. When KF was administered once daily for five consecutive days at a dose of 80 microg/kg per day (400 microg/kg total dose), slightly decreased body weight gain was the primary drug-related effect. Therefore, the no-adverse-effect dose was at or near 80 microg/kg per day (480 microg/m(2) per day). These findings demonstrate that fractionation of a lethal or MTD dose of KF by daily administration for 5 days reduces drug-induced toxicity, and appears to be a viable option for the clinical evaluation of KF for the treatment of cancer.

Protection of marmoset monkeys against EAE by treatment with a murine antibody blocking CD40 (mu5D12).

CD40-CD40 ligand interactions are crucial to cognate interactions between T cells, B cells and antigen-presenting cells (APC), and contribute to non-antigen-specific effector functions of APC in inflammatory disorders. Here we demonstrate that functional blockade of CD40 with an antagonist mouse anti-human CD40 monoclonal antibody (mAb mu5D12) effectively prevents clinical expression of chronic demyelinating experimental autoimmune encephalomyelitis (EAE) in outbred marmoset monkeys, a preclinical model of multiple sclerosis. Anti-CD40 mAb interfered with development of clinical symptoms of marmoset EAE during the treatment period, even when treatment was started several weeks after T cell priming. Magnetic resonance imaging demonstrated inflammatory activity in the brain at initiation of antibody treatment, confirming that treatment interfered with the disease process. Access of therapeutic anti-CD40 to potential sites of action, the secondary lymphoid organs and the brain white matter lesions, was visualized in situ. The present data are the first to demonstrate the clinical potential of blocking APC and effector cell functions using murine antagonist anti-CD40 mAb in the treatment of chronic inflammatory diseases.

Fast Axonal Transport: A Site of Acrylamide Neurotoxicity?

The cellular and molecular site and mode of action of acrylamide (ACR) leading to neurotoxicity has been investigated for four decades, without resolution. Although fast axonal transport compromise has been the central theme for several hypotheses, the results of many studies appear contradictory. Our analysis of the literature suggests that differing experimental designs and parameters of measurement are responsible for these discrepancies. Further investigation has demonstrated consistent inhibition of the quantity of bi-directional fast transport following single ACR exposures. Repeated compromise in fast anterograde transport occurs with each exposure. Modification of neurofilaments, microtubules, energy-generating metabolic enzymes and motor proteins are evaluated as potential sites of action causing the changes in fast transport. Supportive and contradictory data to the hypothesis that deficient delivery of fast-transported proteins to the axon causes, or contributes to, neurotoxicity are critically summarized. A hypothesis of ACR action is presented as a framework for future investigations.