Low expression of miR-203 promoted diabetic nephropathy via increasing TLR4.

OBJECTIVE: To investigate the relationship between microRNA-203 (miR-203) and diabetic nephropathy and its potential mechanism. MATERIALS AND METHODS: The expression of microRNA-203 in mice with diabetic nephropathy and M4200 cells cultured with high glucose was detected by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Toll-like receptor 4 (TLR4), the target gene of microRNA-203, was predicted and screened by bioinformatics method. Real-time quantitative PCR and Western blot were used to detect the endogenous TLR4 level in renal cortex of db/db mice with diabetic nephropathy and glomerular mesangial cells cultured in high glucose or low glucose. The expression of microRNA-203 and TLR4 mRNA were evaluated by RT-PCR after treatment of miR-203 mimics and inhibitor. The protein of TLR4 level was detected by Western blot. Additionally, the proliferation ability of cells was evaluated by Cell Counting Kit-8 (CCK8). The target relationship between microRNA-203 and TLR4 3' UTR was confirmed by luciferase reporter assay RESULTS: The expression of miR-203 was significantly decreased in the kidney of mice with diabetic nephropathy and M4200 cells cultured in high glucose. On the contrary, TLR4 expression was significantly increased. Results of in vitro experiments showed that miR-203 could bind to 3'UTR region of TLR4. Overexpression of microRNA-203 significantly decreased the levels of TLR4 mRNA and protein. Meanwhile, low expression of miR-203 leaded to increased TLR4 expression, resulting in an enhanced proliferation of M4200 cells. CONCLUSIONS: The downregulation of microRNA-203 leaded to an increased level of TLR4, thus promoting proliferation of M4200 cells in the pathogenesis of diabetic nephropathy.

Age-related changes in the performance of forward reach.

Aging is widely considered to be associated with limited balance capacity. It is not clear if forward reach ability is also affected by aging. The purpose of this study was to determine if aging was associated with reduced ability of forward reach or changes in movement patterns. Thirty-three young and 31 older adults were instructed to reach forward as far as possible without losing balance. A motion analysis system was used to record the body kinematics to calculate the joint angle and estimate the motion of center of mass (COM) using a five-segment model. Reach distance (measured from the finger marker), COM displacement, and the distance that the COM exceeded the 2nd toe marker (COM-toe) were used to represent reach performance. The movement patterns were classified as hip, ankle or mixed strategies based upon joint kinematics. It was found that the initial location of the COM was significantly more anterior in the older adults. Older adults were found to have significantly smaller COM displacement and greater hip flexion, but did not differ from young adults in reach distance or COM-toe. Older adults overwhelmingly adopted a hip strategy, but none adopted an ankle strategy. The distribution of the different strategies also differed significantly between groups. These findings suggest that aging appears to be associated with modifications in movement patterns, but not necessarily with a reduction in the ability to approach the boundary of stability. Clinically, balance training for older adults may include the exploration and instruction of atypical movement patterns.

Determinants of pressure wave reflection: characterization by the transit time-independent reflected wave amplitude.

The effects of pressure wave reflection have been incompletely described by the central augmentation index (cAI) and augmented pressure (Pa). We therefore investigated the determinants of amplitude of the reflected wave (Pb), which is independent of the reflected wave transit time (RWTT) and has been shown to predict cardiovascular mortality in the general population. A total of 180 (117 men, mean age 68 years old) patients were recruited. Carotid pressure waveforms derived by tonometry at baseline and 3 min after administration of sublingual nitroglycerin (NTG) were calibrated and then decomposed into the forward and backward waves to yield Pb. The ratio of pre-ejection period/ejection time (PEP/ET) was measured. By stepwise multivariate analysis, independent determinants of Pb included brachial mean blood pressure (ß=0.56, P<0.001), heart rate (ß=-0.29, P<0.001), age (ß=0.20, P<0.001), PEP/ET (ß=-0.16, P=0.004) and height (ß=-0.13, P=0.018). RWTT, body mass index and sex were significant independent determinants of Pa and cAI but did not contribute to Pb. Change of Pb but not Pa or cAI significantly predicted the changes of carotid systolic (r=0.550, P<0.001) and pulse pressure (r=0.618, P<0.001) after NTG. In conclusion, determinants of Pb differ from those of cAI and Pa. Pb is independent of sex and RWTT.

Association between sensorimotor function and forward reach in patients with diabetes.

Deterioration in the function of the sensorimotor system is often seen in patients with diabetes and could be related to balance impairments. The purpose of this study was to determine the association between sensorimotor function and forward reach ability in patients with diabetes. Thirty-one patients with Type 2 diabetes went through a monofilament test of plantar touch-pressure threshold, an ankle joint reposition test for joint position sense, and a series of strength tests of the lower leg. These patients also performed the forward reach test in standing to measure the reach distance and displacement of the center of mass (COM), using a motion analysis system. Correlational and regressional analyses were conducted to determine the association between sensorimotor and balance parameters. It was found that greater reach distance and COM displacement were significantly correlated with lower plantar touch-pressure threshold and greater plantarflexion strength. Regression analysis showed that after controlling the variance in the subject characteristics, plantar touch-pressure threshold was a significant predictor for reach distance and COM displacement, while plantarflexion strength was also a significant predictor for COM displacement. These findings highlight the importance of the assessment of plantar sensitivity and the need for detailed balance or fall risk assessment for patients with impaired plantar insensitivity.

Mifepristone acts as progesterone antagonist of non-genomic responses but inhibits phytohemagglutinin-induced proliferation in human T cells.

BACKGROUND: Progesterone is an endogenous immunomodulator that suppresses T cell activation during pregnancy. The stimulation of membrane progesterone receptors (mPRs) would seem to be the cause of rapid non-genomic responses in human peripheral T cells, such as an elevation of intracellular calcium ([Ca(2+)](i)) and decreased intracellular pH (pH(i)). Mifepristone (RU486) produces mixed agonist/antagonist effects on immune cells compared with progesterone. We explored whether RU486 is an antagonist to mPRs and can block rapid non-genomic responses and the induction by phytohemagglutinin (PHA) of cell proliferation. METHODS: Human male peripheral T cell responses in terms of pH(i) and [Ca(2+)](i) changes were measured using the fluorescent dyes, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) and fura-2, respectively. Expression of mPR mRNA was determined by RT-PCR analysis. Cell proliferation and cell toxicity were determined by [(3)H]-thymidine incorporation and MTT assay, respectively. RESULTS: The mRNAs of mPRalpha, mPRbeta and mPRgamma were expressed in T cells. RU486 blocked progesterone-mediated rapid responses including, the [Ca(2+)](i) increase and pH(i) decrease, in a dose related manner. RU486 did not block, but enhanced, the inhibitory effect of progesterone on PHA induced cell proliferation. RU486 alone inhibited proliferation induced by PHA and at >25 microM seems to be cytotoxic against resting T cells (P < 0.01). CONCLUSIONS: RU486 is antagonistic to the rapid mPR-mediated non-genomic responses, but is synergistic with progesterone with respect to the inhibition of PHA-induced cell proliferation. Our findings shine new light on RU486's clinical application and how this relates to the non-genomic rapid physiological responses caused by progesterone.

Synergic CSE1L/CAS, TNFR-1, and p53 apoptotic pathways in combined interferon-gamma/adriamycin-induced apoptosis of Hep G2 hepatoma cells.

Many cancers are chemotherapy-resistant. Chemotherapy combined with immunotherapy offers a potential avenue for the treatment of chemotherapy-resistant cancers. In this study, we investigated the apoptotic pathways induced by combined interferon-gamma/adriamycin treatment in Hep G2 cells. Our data showed that Hep G2 cells treated with combined interferon-gamma/adriamycin enhanced cell apoptosis in comparison with that of cells treated with adriamycin. Interferon-y increased TNFR-1, CSE1L/CAS (cellular apoptosis susceptibility protein), Bax, and Bad levels. Adriamycin increased p53 and Bax, but not TNFR- 1 and CAS levels. Interferon-y did not increase p53 accumulation; nevertheless it enhanced adriamycin-induced p53 accumulation. Overexpression of IRF-1 augmented the combined interferon-gamma/adriamycin-induced p53 accumulation. Interferon-gamma co-treatment increased the stability of p53 protein induced by adriamycin. Our data suggest that TNF-gamma may greatly enhance the combined interferon-gamma/chemotherapeutic drug-induced apoptosis of cancers. Our findings also indicate that CAS, TN-FR-1, p53, Bax, and Bad may be the targets for the interferon-y-based chemo-immunotherapy of the chemotherapy-resistant cancers.

CSE1/CAS overexpression inhibits the tumorigenicity of HT-29 colon cancer cells.

We previously reported that CSE1/CAS (CAS) overexpression in HT-29 human colon cancer cells enhances the formation of the E-cadherin/beta-catenin complex, stimulates intercellular junction formation, and stimulates polarization of HT-29 cells. Since both E-cadherin/beta-catenin interaction and epithelial cell polarization are critically related to the tumorigenicity of carcinoma cells, we studied the role of CAS in the tumorigenicity of HT-29 colon carcinoma cells. CAS overexpression in HT-29 cells decreased the intercellular gaps and increased the compactness of cell colonies. Our results show that CAS expression inhibited migration and growth of HT-29 cancer cells. In the soft agar anchorage-independent growth assays, CAS overexpression inhibited the colony size of HT-29 cells by 74%, and inhibited colony formation number of HT-29 cells by 38%. CAS overexpression also inhibited the growth of HT-29 cells in nude mice. Our results indicate that CAS inhibits the tumorigenicity of HT-29 human colon cancer cells and, thus, it is worthwhile to further study CAS's possible role in the control of human colon cancer.

IRF-1-mediated CAS expression enhances interferon-gamma-induced apoptosis of HT-29 colon adenocarcinoma cells.

The expression of CAS is reported to be upregulated in a variety of human tumor cells, and such expression correlates with the development of tumors. CAS also plays a role in apoptosis. We investigated whether CAS expression affects the susceptibility of tumor cells to IFN-gamma-induced apoptosis. Our data show that IFN-gamma treatment induces CAS expression in HT-29 tumor cells. IFN-gamma-induced gene expression is primarily mediated by the transcriptional factor, IRF-1. Our data show that IRF-1 mediates IFN-gamma-induced CAS expression. Transfection of HT-29 cells with CAS expression vector did not induce apoptosis of cells; nevertheless, CAS overexpression greatly enhanced IFN-gamma-induced apoptosis of cells. CPP32 is regarded as one of the central apoptosis executioner molecules. CAS overexpression enhances IFN-gamma-induced CPP32 expression. These results indicate that tumor cells highly expressing CAS may be more susceptible to apoptosis induced by reagents that are capable of inducing CAS expression. Thus, CAS may be a target for the elimination of tumors.

Aspirin inhibits matrix metalloproteinase-2 activity, increases E-cadherin production, and inhibits in vitro invasion of tumor cells.

Aspirin (acetylsalicylic acid) is a widely used anti-inflammatory drug. Recently, aspirin was shown to reduce the risk of development of cancer and mortality from it. Tumor metastasis is the most important cause of cancer death. The aim of the present study was to investigate if aspirin affects the invasion of cancer cells. Matrix metalloproteinases (MMPs) and cell adhesion molecules play important roles in the modulation of tumor invasion. Gelatin-based zymography assay showed that aspirin inhibited MMP-2 activity of SK-Hep-1 cancer cells. Matrigel-based chemoinvasion assay showed that aspirin inhibited in vitro invasion of SK-Hep-1 cancer cells. Aspirin treatment also increased the production of the cell adhesion molecule, E-cadherin, in Hep G2 cancer cells. Aspirin is a cyclooxygenase (COX) inhibitor. Treatment of cells with another COX inhibitor, sulindac, also inhibited MMP-2 activity and increased E-cadherin production of cells. These results indicate that aspirin can modulate both MMP-2 and E-cadherin production and therein may possess antimetastatic effect.

Proton-lithium binding behavior of tris(2-((pyrid-2-ylmethyl)uredio)ethyl)amine.

Tris(2-((pyrid-2-ylmethyl)uredio)ethyl)amine (2) and its perchlorate salt, 2.HClO(4), bind with Li+ in nitromethane in a 1:1 fashion. The stability constants of K(Li+) and K(H)(Li+) were found to be 112 +/- 25 and 130 +/- 30 M(-)(1) in CD(3)NO(2), respectively. Formation of the 1:1 complexes were further evidenced by electrospray ionization mass spectrometry (ESI-MS). The slight increase, or at least the same order of magnitude, of K(H)(Li+) compared to K(Li+) points to a remarkable preorganization of the protonated podand in 2.HClO(4), that essentially overcomes the increased Columbic repulsion occurring on complexation to Li+.

Methyl methanesulfonate and hydrogen peroxide differentially regulate p53 accumulation in hepatoblastoma cells.

Genotoxic chemicals not only damage cellular DNA, but may also induce cell apoptosis if they are lethal to the cell. p53, Bcl-2 and Bax play important roles in the regulation of genotoxic chemical induced cell apoptosis. Since the mechanisms by which cellular DNA damaged by different DNA-damaging chemicals may not be the same, we studied the involvement of p53, Bcl-2 and Bax in apoptosis induced by methyl methanesulfonate (MMS) and hydrogen peroxide (H2O2). H2O2 damages DNA by free radical generation and MMS damages DNA by DNA methylation. At non-lethal doses, both H2O2 and MMS induced high level of p53 protein accumulation. Nevertheless, while the amount of p53 protein increased with the dose of MMS and the occurrence of apoptotic cell death events, H2O2 doses that induce cell apoptosis attenuated the p53 protein accumulation level. Lethal MMS treatment also increased Bax, but not Bcl-2 expression, whereas in H2O2 induced apoptosis, the level of both Bcl-2 and Bax declined. These results indicate that toxic chemicals differentially regulate the accumulation of p53 protein. Thus, the pathways of toxic chemicals induced cell apoptosis are different and independent.

Factors related to adolescents' perceptions of treatment outcomes in an adolescent health clinic.

To determine factors related to adolescents' perceived treatment outcomes of their health problems in an adolescent health clinic located at a college hospital, 246 adolescent patients between the ages of 11 and 21 who visited the clinic twice or more during the period January 1994 to December 1995 were included in this study. Information concerning adolescents' sociodemographic characteristics, family function, office visits and health problems of first visits was collected by reviewing subjects' medical and other clinic-related records. In addition, a structured questionnaire was mailed to assess subjects' satisfaction with physicians and the environment and services provided by the clinic as well as their perceived treatment outcomes. 148 patients completed the questionnaire, a response rate of 60.2%. Most of the respondents were in late-stage adolescence (71.0%) and were in school (71.0%). About half of respondents had normal family function, while the other half had moderate or severe family dysfunction. Most of the health problems of respondents were acute (64.2%) and were biological (76.4%) conditions. Most of the respondents were satisfied with the various characteristics of physicians except confidentiality emphasized by the physicians, while many fewer respondents were satisfied with the environment and services provided by the clinic. Family function, physicians' respect toward the adolescents, and the adolescents' satisfaction with the services provided in general were the factors significantly related to adolescents' perceived treatment outcomes based on a stepwise, multiple logistic regression analysis. We conclude that efforts to provided could result in better adolescent perceived treatment outcomes.

Involvement of serine proteinase in infectious pancreatic necrosis virus capsid protein maturation and NS proteinase cleavage in CHSE-214 cells.

An investigation of virus-specific protein maturation in infectious pancreatic necrosis virus (IPNV) infected Chinook salmon embryo cells (CHSE-214) was undertaken. The precursor protein (pVP2-1) of the major mature capsid protein (VP2) was processed sequentially from pVP2-1 to pVP2-2 and VP2. Experiments using serine proteinase inhibitors showed that the maturation of the VP2 was blocked in the pVP2-1 post-translational cleavage steps. A protinin, a potent proteinase inhibitor, at 800 µg ml(-1) blocked pVP2-2 to VP2 and the cleavage of VP4 (28 kDa) to VP4-1 (25 kDa). Therefore, our data showed that the maturation of the capsid protein (VP2) and cleavage of VP4 (NS proteinase) can be blocked by serine proteinase inhibitors.

Characteristics of adolescent patients and their health problems at an adolescent health clinic.

In Taiwan, there has been an increase in biopsychosocial morbidity among adolescents in the past ten years, but neither an adolescent health care delivery system nor a subspecialty training program in adolescent medicine has been hitherto established. To better understand the characteristics of adolescent patients and their health problems, the sociodemography and health problems of all adolescent patients aged 11 to 21 years who visited the Adolescent Health Clinic twice or more during January, 1994 and December, 1995 at a college hospital, were analysed by reviewing patients' medical records and the adolescent registration records. There were totally 264 adolescent patients with an average age of 18.7 +/- 3.4 years. The number of female adolescents involved in the study (139 persons or 52.7%) was little more than that of male adolescents. Most of the adolescent patients (67.4%) were in late stage of adolescence; in addition, most of them (81.4%) were students. During the 2-year study period, there was a total of 350 independent health problems among 957 office visits made by the 264 patients, averaging 1.3 problems and 2.7 office visits per patient and per problem, respectively. Supplementary classification, mental disorders, and respiratory system diseases were the three leading disease classifications, occupying 66.6% of all disease classifications. General medical examination, upper respiratory tract infection, and immunization were more common among all individual diagnoses. Regarding gender difference in the encountered health problems, hepatitis B, office visiting for counseling, and conductive disorder, were more prominent in the male adolescents, whereas upper respiratory tract infection, goiter, acne vulgaris, peptic ulcer disease, migraine, and eating disorders were more common in the female adolescents. Moreover, the patients in the different stages of adolescence had their unique health problems. It was concluded that there were variations in the health problems encountered among adolescents of different genders and of the different stages of adolescence.

Vaninolol: a new selective beta 1-adrenoceptor antagonist derived from vanillin.

The beta-adrenoceptor blocking properties of vaninolol ((+/-)4-[4'-(2-hydroxy-3-tert-butyl-aminopropoxy)-3'-methoxyphenyl]- 3-buten-2-one), derived from vanillin, were first investigated under in vivo and in vitro conditions. Vaninolol (0.1, 0.5, 1.0 mg/kg, i.v.), as well as propranolol, produced a dose-dependent bradycardia response and a sustained pressor action in urethane-anesthetized normotensive rats. Vaninolol inhibited the tachycardia effects induced by (-)isoproterenol, but had no blocking effect on the arterial pressor responses induced by phenylephrine. These findings suggested that vaninolol possessed beta-adrenergic blocking activity, but was without alpha-adrenergic blocking activity. In isolated guinea-pig tissues, vaninolol antagonized (-)isoproterenol-induced positive inotropic and chronotropic effects of the atria and tracheal relaxation responses in a concentration-dependent manner. The parallel shift to the right of the concentration-response curve of (-)isoproterenol suggested that vaninolol was a beta-adrenoceptor competitive antagonist. The effect of vaninolol was more potent on the atria than on tracheal tissues, indicating it had some beta 1-adrenoceptor selectivity. On the other hand, the order of the hydrophilicity was atenolol >> vaninolol > propranolol. In addition, vaninolol had a mild direct cardiac depression at high concentrations and was without intrinsic sympathomimetic activity (ISA). Furthermore, binding characteristics of vaninolol and other beta-adrenoceptor antagonists were evaluated in [3H]dihydroalprenolol binding to guinea-pig ventricular membranes. The order of potency of beta-adrenoceptor antagonists in competing for the binding sites was (-)propranolol >> vaninolol > or = atenolol. In conclusion, vaninolol was found to be a selective beta 1-adrenoceptor antagonist with relatively low lipophilicity in comparison with propranolol, devoid of ISA, and had a mild myocardial depressant effect.

Characterization of beta-adrenoceptors of tilapia erythrocytes with hydrophobic and hydrophilic radioligands.

Tilapia are widely used for to investigate stress physiology, and beta-adrenoceptors in fish erythrocytes play important roles in response to hypoxia and other environmental stresses. As the beta-adrenoceptor in tilapia erythrocytes has never been investigated, we characterized beta-adrenoceptors in intact red blood cells of a tilapia (Oreochromis mossambicus) in this study by radioligand binding assay with a hydrophobic beta-adrenoceptor antagonist, 1-[4,6-propyl-3H]dihydroalprenolol ([3H]DHA), and a hydrophilic ligand (-)-4-(3-t-butylamino-2-hydroxypropoxy)-[5,7-3H] benzimidazol-2-one ([3H]CGP-12177). The equilibrium dissociation constant (KD) for [3H]CGP-12177 calculated from kinetic experiments (KD = 5.8 +/- 4.8 nM) was comparable to that obtained from Scatchard analysis (KD = 1.22 +/- 0.18 nM). However, the KD for [3H]DHA obtained from kinetic studies (KD = 0.41 +/- 0.12 nM) was much smaller than that from a Scatchard plot (KD = 7.8 +/- 1.6 nM). The hydrophobic [3H]DHA bound to two sites (KD = 7.8 nM, Bmax = 12,500 sites/cell and KD = 77.4 nM, Bmax = 70,550 sites/cell), whereas the hydrophilic [3H]CGP-12177 bound to one site (KD = 1.2 nM, Bmax = 1,900 sites/cell). The results indicate that high-affinity beta-adrenoceptors are located both on the surface of and inside tilapia erythrocytes, and low-affinity receptors exist only inside erythrocytes.

Functional expression of rat M5 muscarinic acetylcholine receptor in yeast.

We have produced the rat M5 muscarinic acetylcholine receptor, an integral membrane protein, in the yeast Saccharomyces cerevisiae. This was achieved by placing an M5 gene in the yeast vector under the control of the yeast alpha-factor promoter and leader sequence. Northern blotting revealed the presence of M5 transcripts in yeast transformed with the M5 plasmid constructs. Crude extract prepared from the transformant yeasts showed saturable binding of the muscarinic antagonist [3H]-N-methyl scopolamine ([3H]NMS) with a kd of 22.77 nM and Bmax of 134.76 fmole per mg protein. Results deduced from saturation binding assay of intact cell demonstrated clearly that the M5 receptor was translocated across the membrane of the endoplasmic reticulum using the secretion signal on alpha-leader sequence and its binding site was still functional. Yeast expressing M5 receptor did not exhibit cell-cycle arrest in the presence of carbachol, a acetylcholine agonist, indicating that the recombinant M5 receptor could not couple directly to the endogenous yeast pheromone signaling G-protein.

Cellular responses to stimulation of the M5 muscarinic acetylcholine receptor as seen in murine L cells.

The membrane signaling properties of the neuronal type-5 muscarinic acetylcholine receptor (M5 AChR) as expressed in murine L cells were studied. Recipient Ltk- cells responded to ATP acting through a P2-purinergic receptor by increasing phosphoinositide hydrolysis 2-fold but were unresponsive to 17 receptor agonists that are stimulatory in other cells. L cells expressing the M5 AChR responded to carbachol (CCh) with an approximately 20-fold increase in phospholipase C activity, mobilization of Ca2+ from endogenous stores, causing a transient peak increase in the intracellular concentration of Ca2+ ([Ca2+]i), influx of extracellular Ca2+, causing a sustained increase in [Ca2+]i dependent on extracellular Ca2+, and release of [3H]arachidonic acid from prelabeled cells, without altering resting or prostaglandin E1-elevated intracellular cAMP levels. None of the effects of the M5 AChR were inhibited by pertussis toxin. The regulation of L cell [Ca2+]i was studied further. ATP had the same effects as CCh and the two agonists acted on a shared intracellular pool of Ca2+. The peak and sustained [Ca2+]i increases were reduced by cholera toxin and forskolin, neither of which altered significantly phosphoinositide hydrolysis. This is consistent with interference with the action of inositol 1,4,5-trisphosphate (IP3) through cAMP-mediated phosphorylation and suggests a continued involvement of IP3 during the sustained phase of [Ca+]i increases. The temporal pattern of the sustained [Ca2+]i increase differed whether elicited by CCh or ATP, and was enhanced in pertussis toxin-treated cells. This is consistent with existence of a kinetic control of the sustained [Ca2+]i change by a receptor-G protein-dependent mechanism independent of the IP3 effector site(s) (e.g. pulsatile activation of phospholipase C and/or pulsatile activation of a receptor/G protein-operated plasma membrane Ca2+ channel). Thus, the non-excitable L cell may be a good model for studying [Ca2+]i regulations, as may occur in other nonexcitable cells of which established cell lines do not exist, and for studying of receptors that as yet cannot be studied in their natural environment.

Molecular cloning and expression of a fifth muscarinic acetylcholine receptor.

A cDNA of 2149 base pairs with an incomplete open reading frame (ORF) encoding amino acids 1-516 of a 531-amino acid protein highly homologous to muscarinic receptors was cloned from a rat brain cDNA library. The complete ORF was then deduced from a DNA fragment cloned from a rat genomic library. This ORF was subcloned into the eukaryotic expression vector p91023(B) under control of the adenovirus major late promoter and co-transfected with the thymidine kinase selection marker into muscarinic receptor-negative, thymidine kinase-negative murine L cells. Stable transformants were selected and tested for acquisition of muscarinic receptors by following appearance of specific binding sites for the muscarinic ligand [3H] N-methylscopolamine. Two cell lines, LM5.36 and LM5.40, were cloned and shown to express typical muscarinic receptor sites, thus confirming that the newly cloned ORF encodes a muscarinic receptor, the rat M5 muscarinic acetylcholine receptor. Tests for activities showed it to stimulate phosphoinositide hydrolysis in intact cells, without affecting positively or negatively adenylyl cyclase activity. The M5 receptor contains two putative glycosylation sites at its amino terminus and, based on hydropathicity analysis, is predicted to span the plasma membrane seven times. Like 17 other receptors of this class, the M5 receptor has 19 conserved amino acids, among which are 4 prolines located in the 4th, 5th, 6th, and 7th predicted transmembrane regions, conferring possible bends to these helices, and 2 cysteines, one in the 1st and the other in the 2nd extracellular loop, possibly providing for a disulfide bond. Similarity in amino acid composition and in patterns of antagonist binding and biologic effects suggest the M5 receptor to be M1-like.