Biomedical subjects

Kevin Lee

Publications and source records attributed to Kevin Lee.

17 recordsLinked to original sources

PubMed2006

The putative transmembrane segment 7 of human organic anion transporter hOAT1 dictates transporter substrate binding and stability.

Human organic anion transporter hOAT1 plays a critical role in the body disposition of clinically important drugs. We examined the role of the putative transmembrane segment (TM) 7 in the function of hOAT1. Each residue within putative TM7 was replaced by alanine, and the uptake of para-aminohippurate was studied in cells expressing the mutants. We discovered four critical amino acid residues: Trp-346, Thr-349, Tyr-353, and Tyr-354. Substitution of Tyr-353 and Tyr-354 with alanine led to the loss of transport activity without affecting the surface expression of the transporter, whereas substitution of Trp-346 and Thr-349 with alanine lead to the loss of the total expression of the transporter. The effect of side chains of Tyr-353 and Tyr-354 on transporter functions were further evaluated by replacing these residues with Phe or Trp. Among all the mutants studied (Y353W, Y353F, Y354W, and Y354F), only mutant Y353F regained 30% transport activity, which was lost from replacement of Tyr-353 with alanine, suggesting that both the -OH group and the size of the side chain at positions 353 and 354 are critical for maintaining the full transport activity. To investigate the mechanisms underlying the loss of total protein expression when Trp-346 and Thr-349 were replaced with alanine, mutant-expressing cells were treated with lysosomal or proteasomal inhibitors. Our results showed that only proteasomal inhibitors resulted in the accumulation of mutant proteins, indicating that proteasome is involved in the degradation of the mutant transporters. Therefore, Trp-346 and Thr-349 are critically involved in the stability of the transporter.

Alanine↗

PubMed2006

Dual role of 5-HT3 receptors in a rat model of delayed stress-induced visceral hyperalgesia.

Despite its beneficial effect in IBS patients, the mechanism of action of the 5-HT3 receptor (5-HT3R) antagonist alosetron is still incompletely understood. We aimed to characterize the effect and site(s) of action in a model of stress-induced sensitization of visceral nociception in rats. Adult male Wistar rats were equipped for recording of visceromotor response (VMR) to phasic colorectal distension (CRD; 10-60 mmHg). VMR to CRD was recorded 24 h after an acute session of water avoidance (WA) stress (post-WA). Baseline and post-WA responses were measured in rats exposed to WA or sham-WA, treated with alosetron at 0.3 mg/kg subcutaneously (s.c.) 25 nmol intrathecally (i.t.) or vehicle before post-WA CRD. Some rats were treated with capsaicin/vehicle on the cervical vagus nerve and received alosetron (0.3 mg/kg, s.c.) 15 min before post-WA CRD. WA stress led to visceral hyperalgesia 24 h later. Alosetron (0.3 mg/kg, s.c.), failed to inhibit WA-induced exacerbation of VMR to CRD. Stress-induced visceral hyperalgesia was abolished when alosetron was injected intrathecally (P<0.05) in intact rats or subcutaneously (0.3 mg/kg) in capsaicin-pretreated animals (P<0.05). Capsaicin-pretreatment did not affect the exacerbating effect of stress on visceral sensitivity. Alosetron had no inhibitory effect on normal visceral pain responses when administered subcutaneously or intrathecally. We demonstrated that 5-HT3Rs on central terminals of spinal afferents are engaged in the facilitatory effect of stress on visceral sensory information processing. In addition, we showed that stress-induced sensitization of visceral nociception is independent of 5-HT3R activation on vagal afferents.

Acute Disease↗

PubMed2006

5-HT in the enteric nervous system: gut function and neuropharmacology.

In recent times, the perception of functional gastrointestinal disorders such as irritable bowel syndrome (IBS) has shifted fundamentally. Such disorders are now thought of as serious diseases characterized by perturbations in the neuronal regulation of gastrointestinal function. The concept of visceral hypersensitivity, the characterization of neuronal networks in the 'brain-gut axis' and the identification of several novel 5-HT-mediated mechanisms have contributed to this shift. Here, we review how some of the more promising of these new mechanisms (e.g. those involving 5-HT transporters and the 5-HT(2B), 5-HT(7) and putative 5-HT(1p) receptors) might lead to a range of second-generation therapies that could revolutionize the treatment of functional gastrointestinal disorders, particularly IBS.

Animals↗

PubMed2006

Pain.

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Animals↗

PubMed2006

Indian hedgehog is a major mediator of progesterone signaling in the mouse uterus.

The hedgehog family of morphogens are regulators of cell proliferation, differentiation and cell-cell communication. These morphogens have been shown to have important roles in organogenesis, spermatogenesis, stem cell maintenance and oncogenesis. Indian hedgehog (encoded by Ihh) has been shown to be expressed in the uterine epithelium under the control of the steroid hormone, progesterone. Although in vivo and in vitro studies have shown that progesterone achieves its effects on uterine function through epithelial-stromal cross-talk, molecular mediator(s) for this cellular communication pathway have not been elucidated. Using new experimental approaches that ablate Ihh specifically in Pgr-positive uterine cells of the mouse, we demonstrate that Ihh is an essential mediator of Pgr action in the uterus, and expression of this factor is critical in mediating the communication between the uterine epithelium and stroma required for embryo implantation.

Animals↗

PubMed2005

Histamine excites neonatal rat sympathetic preganglionic neurons in vitro via activation of H1 receptors.

The role of histamine in regulating excitability of sympathetic preganglionic neurons (SPNs) and the expression of histamine receptor mRNA in SPNs was investigated using whole-cell patch-clamp electrophysiological recording techniques combined with single-cell reverse transcriptase polymerase chain reaction (RT-PCR) in transverse neonatal rat spinal cord slices. Bath application of histamine (100 microM) or the H1 receptor agonist histamine trifluoromethyl toluidide dimaleate (HTMT; 10 microM) induced membrane depolarization associated with a decrease in membrane conductance in the majority (70%) of SPNs tested, via activation of postsynaptic H1 receptors negatively coupled to one or more unidentified K+ conductances. Histamine and HTMT application also induced or increased the amplitude and/or frequency of membrane potential oscillations in electrotonically coupled SPNs. The H2 receptor agonist dimaprit (10 microM) or the H3 receptor agonist imetit (100 nM) were without significant effect on the membrane properties of SPNs. Histamine responses were sensitive to the H1 receptor antagonist triprolidine (10 microM) and the nonselective potassium channel blocker barium (1 mM) but were unaffected by the H2 receptor antagonist tiotidine (10 microM) and the H3 receptor antagonist, clobenpropit (5 microM). Single cell RT-PCR revealed mRNA expression for H1 receptors in 75% of SPNs tested, with no expression of mRNA for H2, H3, or H4 receptors. These data represent the first demonstration of H1 receptor expression in SPNs and suggest that histamine acts to regulate excitability of these neurons via a direct postsynaptic effect on H1 receptors.

Action Potentials↗

PubMed2005

Impact of race, age, income, and residence on prostate cancer knowledge, screening behavior, and health maintenance in siblings of patients with prostate cancer.

OBJECTIVE: This study evaluates self-reported changes in knowledge of prostate cancer (CaP), CaP screening behavior and other health-related activities in men whose brothers have been diagnosed with prostate cancer and to characterize those demographic subgroups of siblings at particular risk of failure to change their behavior. METHODS: 112 men were surveyed to self-assess their knowledge of CaP both before and after their brother's diagnosis. In addition, siblings were questioned with regard to other health-related behaviors before and after their brother's diagnosis. Demographic characteristics including age, race, income, and area of residence were also reported. Results were analyzed to determine how a brother's diagnosis with CaP affects changes in prostate cancer knowledge, screening behavior and other health related activities in these high-risk patients. RESULTS: The impact of a brother's diagnosis of CaP affects only 40% of siblings with regard to CaP knowledge. This knowledge appears to translate into increased screening behavior and other-health-related activity in these men. Unfortunately, more than half of men have no change in CaP knowledge and correspondingly no change in screening behavior. Siblings who are older, earn less, and live in rural areas have a lower baseline knowledge of CaP and are less likely to improve their self-assessed knowledge. On regression analysis, correlates of improvement in CaP knowledge included (1) those discussing with their primary physician (OR=6.6), (2) Caucasians (OR=2.7) and (3) non-smokers (OR=3.1). Improvements in prostate cancer knowledge were found to be predictive of subsequent participation in CaP screening and annual physical exams. CONCLUSIONS: Increased attention should be paid to siblings of CaP patients in efforts to improve their knowledge and thereby participation in screening as these patients are at an increased risk of development of this disease. Efforts should be made to educate siblings who are older, of lower income, African American, and rural residents with regard to CaP, as these subgroups appear less likely to change their knowledge and screening behavior despite their sibling's diagnosis.

Age Factors↗

PubMed2005

Characterization of retinal damage in the episcleral vein cauterization rat glaucoma model.

Episcleral vein cauterization (EVC) is used in rats to generate a glaucoma model with high intraocular pressure (IOP). The long-term retinal damage in this glaucoma model, however, has not been accurately quantified. We report the location and amount of retinal ganglion cell (RGC) damage caused by (EVC) induced IOP elevation in two rat strains. IOP was raised in one eye of Wistar (N = 5) and Brown-Norway(B-N)(N = 7) rats by EVC and monitored monthly until IOP in contralateral eyes equalized at 5 months post-surgery. Animals were maintained for 3.5-4.5 additional months. B-N rats (N = 7) that had no EVC served as controls for this strain. Scotopic flash ERGs were recorded at baseline and just prior to euthanasia. Automated counts of all retrogradely labeled RGCs in retinal flat-mounts were determined and compared between contralateral eyes. RGC density maps were constructed and RGC size distribution was determined. Oscillatory potentials in the group of eyes which had elevated IOP were decreased at the time of euthanasia, when IOP had returned to normal. The group of normal B-N rats had similar RGC counts between contralateral eyes. In the experimental group the mean number of RGCs was not significantly different between control and experimental eyes, but 1 of 5 Wistar and 2 of 7 B-N experimental eyes had at least 30% fewer RGCs than contralateral control eyes. Total retinal area in B-N experimental eyes was higher compared to contralateral eyes. Cumulative IOP exposure of the experimental eyes was modestly correlated with RGC loss while oscillatory potentials appeared to be inversely related to RGC loss. In retinas with extensive (> 30% RGC loss) but not complete damage, smaller cells were preserved better than larger ones. The above results indicate that RGC loss in both Wistar and B-N strains is variable after a prolonged elevation of IOP via EVC. Such variability despite equivalent IOP levels and ERG abnormalities, suggests unknown factors that can protect IOP-stressed RGCs. Identification and enhancement of such factors could prove useful for glaucoma therapy.

Animals↗

PubMed2005

Haploinsufficiency of chicken ovalbumin upstream promoter transcription factor II in female reproduction.

The chicken ovalbumin upstream promoter transcription factor II, COUP-TFII, is a member of the orphan nuclear receptor transcription factor family. Genetic ablation of COUP-TFII results in early embryonic lethality and demonstrates that this gene is required for cardiac and vascular development. Expression of COUP-TFII persists throughout postnatal life in various tissues including the female reproductive tract. However, the physiological function of COUP-TFII in female reproduction has not been extensively analyzed. Here, we provide phenotypic evidences that haploinsufficiency of COUP-TFII in mice demonstrates an important role of COUP-TFII for normal female reproduction. COUP-TFII +/- females show significantly reduced fecundity, irregular estrus cycles, delayed puberty, and retarded postnatal growth. Analysis of the reduced fertility revealed that although ovarian function was normal with respect to ovulation, the ovaries have reduced ability to synthesize progesterone in response to exogenous gonadotropins. This reduction is due to the reduction of the expression of steroidogenic enzymes important for progesterone synthesis and the reduction of vascularization in COUP-TFII heterozygotes. Analysis of uterine function demonstrated a reduced response to an experimentally induced decidual cell reaction indicating that the ability of the uterus to support embryo implantation was reduced. Taken together, our data show global impact of gene dosage effects of COUP-TFII on female postnatal life and indicates requirement of COUP-TFII in normal female reproduction, in particular for uterine endometrial functions during the peri-implantation period.

Animals↗

PubMed2004

Orexigen-sensitive NPY/AgRP pacemaker neurons in the hypothalamic arcuate nucleus.

The hypothalamic arcuate nucleus (ARC) integrates and responds to satiety and hunger signals and forms the origins of the central neural response to perturbations in energy balance. Here we show that rat ARC neurons containing neuropeptide Y (NPY) and agouti-related protein (AgRP), which are conditional pacemakers, are activated by orexigens and inhibited by the anorexigen leptin. We propose a neuron-specific signaling mechanism through which central and peripheral signals engage the central neural anabolic drive.

4-Aminopyridine↗

PubMed2004

Nitric oxide inhibits NMDA currents in a subpopulation of substantia gelatinosa neurons of the adult rat spinal cord.

In the present study, the actions of nitric oxide (NO) on NMDA mediated excitatory neurotransmission in substantia gelatinosa (SG) neurons of the adult rat spinal cord were investigated. Bath application of the NO donor sodium-nitroso-N-acetylpenicillamine (SNAP) reversibly inhibited NMDA receptor mediated excitatory postsynaptic currents evoked by electrical stimulation of the dorsal root. These effects of SNAP were prevented by pre-incubation with the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy PTIO) or the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one, and were mimicked by the membrane permeable cGMP analogue dibutyryl cGMP. Finally, these effects were shown to be mediated at a postsynaptic level using acutely applied exogenous NMDA. In conclusion, we demonstrate for the first time the inhibition of NMDA mediated excitatory neurotransmission by NO in spinal cord SG neurons, an action mediated at least in part via the activation of guanylate cyclase.

Adaptation, Physiological↗

PubMed2003

Lidocaine block of neonatal Nav1.3 is differentially modulated by co-expression of beta1 and beta3 subunits.

The effects of lidocaine on neonatal Na(v)1.3 (Na(v)1.3n) expressed alone and in combination with beta1 and beta3 subunits in Xenopus oocytes were examined. Lidocaine reversibly inhibited the peak Na(v)1.3n current, shifted the steady-state inactivation curve to hyperpolarized potentials and delayed recovery from inactivation. These effects were attenuated by the co-expression of the beta subunits, with greater attenuating effects being observed in oocytes co-expressing beta1 compared to those co-expressing beta3. Use-dependent block by lidocaine was assessed at 1 Hz train frequency for 60 pulses. Lidocaine caused similar use-dependent block of current amplitude at pulse 60 for Na(v)1.3n and Na(v)1.3n+beta3. In oocytes co-expressing beta1, these use-dependent actions were reduced. In conclusion, the effects of lidocaine on Na(v)1.3n are differentially modulated by beta1 and beta3 subunits. Since these subunits exhibit a complementary distribution, this finding may have importance in our understanding of lidocaine action.

Animals↗

PubMed2003

Orexins induce increased excitability and synchronisation of rat sympathetic preganglionic neurones.

The neuropeptides orexin A and B are synthesised by perifornical and lateral hypothalamic (LH) neurones and exert a profound influence on autonomic sympathetic processes. LH neurones project to spinal areas containing sympathetic preganglionic neurones (SPNs) and therefore may directly modulate sympathetic output. In the present study we examined the possibility that orexinergic inputs from the LH influence SPN activity. Orexin-positive neurones in the LH were labelled with pseudorabies virus injected into the liver of parasympathetically denervated animals and orexin fibres were found adjacent to the soma and dendrites of SPNs. Orexin A or B (10-1000 nM) directly and reversibly depolarised SPNs in spinal cord slices. The response to orexin A was significantly reduced in the presence of the orexin receptor 1 (OX1R) antagonist SB334867A at concentrations of 1-10 micro M. Single cell reverse transcriptase-polymerase chain reaction revealed expression of mRNA for both OX1R and OX2R in the majority of orexin-sensitive SPNs. The orexin-induced depolarisation involved activation of pertussis toxin-sensitive G-proteins and closure of a K+ conductance via a protein kinase A (PKA)-dependent pathway that did not require an increase in intracellular Ca2+. Orexins also induced biphasic subthreshold membrane potential oscillations and synchronised activity between pairs of electrically coupled SPNs. Coupling coefficients and estimated junctional conductances between SPNs were not altered indicating synchronisation is due to activation of previously silent coupled neurones rather than modulation of gap junctions. These findings are consistent with a direct excitation and synchronisation of SPNs by orexinergic neurones that in vivo could increase the frequency and coherence of sympathetic nerve discharges and mediate LH effects on sympathetic components of energy homeostasis and cardiovascular control.

Adrenergic Fibers↗

PubMed2003

Leading the charge--pioneering treatments in the fight against neuropathic pain.

Neuropathic pain, arising from nerve injury, results in a chronic and debilitating form of pain that in the past has been poorly diagnosed and treated. During the past few years, intensive research has resulted in major progress towards understanding the basic mechanisms that contribute to this condition, and the renewed possibility of safe and effective medicines. Indeed, the next few years should see the first fruits of this labour reaching the market place and, with them, the opportunity to assess whether the scientific advances achieved have resulted in significant clinical improvement. In this article, some of the key compounds that will lead this charge and the clinical results obtained so far are discussed.

Anticonvulsants↗

PubMed2003

Emerging antiobesity drugs.

The healthcare burden that the obesity epidemic now poses in highly significant, in part due to increased risk of secondary chronic diseases such as hypertension. A lack of physical activity and high fat diets are major factors contributing to this condition. However, increasingly apparent is the genetic predisposition of individuals and ethnic groups to obesity. Present treatment strategies are currently inadequate and unlikely to have a major effect on the future prevalence of obesity. To slow the obesity epidemic, the source needs to be tackled now through fundamental research into the mechanisms by which obesity is manifest, and education on the risks and how to prevent it. This article will describe current and emerging treatments for obesity and review the recent advances in research that may provide the antiobesity treatments of the future. Research into obesity has escalated at considerable pace, catalysed by the discovery of the obese gene product leptin. Leptin is secreted by adipose tissue and acts via specific receptors in the brain to engage central neural pathways involved in regulating energy homeostasis. Since this discovery, numerous significant advances have been made in our understanding of how the brain integrates and responds to central and peripheral signals involved in maintaining energy homeostasis, and how disruption of these signalling mechanisms can manifest as obesity. As a consequence of these findings, numerous potential sites for therapeutic intervention into this condition have and are materializing. The aim of this review is to highlight current treatment strategies for obesity, recent advances in our understanding of the central neural control of energy balance, and what the authors consider to be the most promising targets for the development of novel antiobesity drugs in the future. Thus, the review focuses on leptin, neuropeptide Y, melanocortin and ghrelin signalling at the level of the CNS, and strategies targeting the sympathetic innervation of fat cells at the periphery.

Animals↗

PubMed2002

Functional and molecular characterization of metabotropic glutamate receptors expressed in rat striatal cholinergic interneurones.

In the present study we have used single-cell RT-PCR in conjunction with electrophysiology to examine the expression and functional properties of metabotropic glutamate receptors (mGluRs) expressed within biochemically identified cholinergic interneurones in the rat striatum. Using single-cell RT-PCR, it was possible to demonstrate the presence of mGluR1, mGluR2, mGluR3, mGluR5 and mGluR7 mRNAs within single cholinergic interneurones. Bath application of the non-selective mGluR agonist (1 S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1 S,3R-ACPD) or the group-I mGluR agonist 3,5-dihydroxyphenylglycine (DHPG) depolarized all cholinergic neurones tested by activation of an inward current at -60 mV. The effects of DHPG were partially inhibited by the mGluR5 selective antagonist 6-methyl-2-(pherazo)-3-pyridinol and by the non-selective group-I antagonist alpha-methyl-4-carboxyphenylglycine but were not mimicked by the group-II and group-III selective mGluR agonists 2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV) and L-2-amino-4-phosphonobutanoate (L-AP4), respectively. Intrastriatal stimulation evoked an excitatory postsynaptic current within cholinergic neurones that was reversibly inhibited by bath application of the group-II and group-III selective mGluR agonists DCG-IV and L-AP4, respectively, via presynaptic actions. In summary, we have identified the mGluRs expressed by striatal cholinergic interneurones and demonstrated that their activation produces modulatory effects via both pre- and postsynaptic mechanisms.

Animals↗

PubMed2002

Effect of theory-based feature correlations on typicality judgments.

In the present study, we examine what types of feature correlations are salient in our conceptual representations. It was hypothesized that of all possible feature pairs, those that are explicitly recognized as correlated (i.e., explicit pairs) and affect typicality judgments are the ones that are more likely theory based than are those that are not explicitly recognized (i.e., implicit pairs). Real-world categories and their properties, taken from Malt and Smith (1984), were examined. We found that explicit pairs had a greater number of asymmetric dependency relations (i.e., one feature depends on the other feature, but not vice versa) and stronger dependency relations than did implicit pairs, which were statistically correlated in the environment but were not recognized as such. In addition, people more often provided specific relation labels for explicit pairs than for implicit pairs; these labels were most often causal relations. Finally, typicality judgments were more affected when explicit correlations were broken than when implicit correlations were broken. It is concluded that in natural categories, feature correlations that are explicitly represented and affect typicality judgments are the ones about which people have theories.

Adult↗