Sociodemographic and psychopathologic predictors of first incidence of DSM-IV substance use, mood and anxiety disorders: results from the Wave 2 National Epidemiologic Survey on Alcohol and Related Conditions.

The objective of this study was to present nationally representative findings on sociodemographic and psychopathologic predictors of first incidence of Diagnostic and Statistical Manual of Mental Disorders, 4th edn (DSM-IV) substance, mood and anxiety disorders using the Wave 2 National Epidemiologic Survey on Alcohol and Related Conditions. One-year incidence rates of DSM-IV substance, mood and anxiety disorders were highest for alcohol abuse (1.02), alcohol dependence (1.70), major depressive disorder (MDD; 1.51) and generalized anxiety disorder (GAD; 1.12). Incidence rates were significantly greater (P<0.01) among men for substance use disorders and greater among women for mood and anxiety disorders except bipolar disorders and social phobia. Age was inversely related to all disorders. Black individuals were at decreased risk of incident alcohol abuse and Hispanic individuals were at decreased risk of GAD. Anxiety disorders at baseline more often predicted incidence of other anxiety disorders than mood disorders. Reciprocal temporal relationships were found between alcohol abuse and dependence, MDD and GAD, and GAD and panic disorder. Borderline and schizotypal personality disorders predicted most incident disorders. Incidence rates of substance, mood and anxiety disorders were comparable to or greater than rates of lung cancer, stroke and cardiovascular disease. The greater incidence of all disorders in the youngest cohort underscores the need for increased vigilance in identifying and treating these disorders among young adults. Strong common factors and unique factors appear to underlie associations between alcohol abuse and dependence, MDD and GAD, and GAD and panic disorder. The major results of this study are discussed with regard to prevention and treatment implications.

Neutral fat hydrolysis and long-chain fatty acid oxidation during anaerobic digestion of slaughterhouse wastewater.

Neutral fat hydrolysis and long-chain fatty acid (LCFA) oxidation rates were determined during the digestion of slaughterhouse wastewater in anaerobic sequencing batch reactors operated at 25 degrees C. The experimental substrate consisted of filtered slaughterhouse wastewater supplemented with pork fat particles at various average initial sizes (D(in)) ranging from 60 to 450 microm. At the D(in) tested, there was no significant particle size effect on the first-order hydrolysis rate. The neutral fat hydrolysis rate averaged 0.63 +/- 0.07 d(-1). LCFA oxidation rate was modelled using a Monod-type equation. The maximum substrate utilization rate (kmax) and the half-saturation concentration (Ks) averaged 164 +/- 37 mg LCFA/L/d and 35 +/- 31 mg LCFA/L, respectively. Pork fat particle degradation was mainly controlled by LCFA oxidation rate and, to a lesser extent, by neutral fat hydrolysis rate. Hydrolysis pretreatment of fat-containing wastewaters and sludges should not substantially accelerate their anaerobic treatment. At a D(in) of 450 microm, fat particles were found to inhibit methane production during the initial 20 h of digestion. Inhibition of methane production in the early phase of digestion was the only significant effect of fat particle size on anaerobic digestion of pork slaughterhouse wastewater. Soluble COD could not be used to determine the rate of lipid hydrolysis due to LCFA adsorption on the biomass.

Treatment and outcome of Taiwanese patients with 6-pyruvoyltetrahydropterin synthase gene mutations.

Ten cases of tetrahydrobiopterin (BH4) deficiency were identified in 1,337,490 newborns screened in a Chinese population in Taiwan. The high incidence of BH4 deficiency in the Taiwanese population may be explained by a founder effect, since all of the patients revealed 6-pyruvoyltetrahydropterin synthase gene mutations, and grouping N52S and P87S mutations together constituted 88.9% of the disease alleles. BH4 supplementation with restriction of high-protein foods gave control of plasma phenylalanine within normal range, and levodopa itself prevented seizure. However, the average intelligence quotient (IQ) score of these patients was only 76 +/- 14 (56-98). Statistically, the age of starting medication, including 5-hydroxytryptophan (5-HTP), was inversely correlated to IQ scores of these patients. We suggest the combination of BH4, levodopa and 5-HTP as the standard protocol to commence the treatment of BH4 deficiency as early as possible, although prenatal brain damage could have existed.

Expression of the Epstein-Barr virus BHRF1 gene, a homologue of Bcl-2, in nasopharyngeal carcinoma tissue.

Epstein-Barr virus (EBV) infection is associated closely with the pathogenesis of nasopharyngeal carcinoma (NPC). The EBV gene product, BHRF1, has been demonstrated in vitro and is structurally and functionally similar to the oncogene bcl-2, that is able to protect cells from programmed cell death. To determine whether the BHRF1 gene is expressed in vivo, BHRF1 mRNA or protein were sought in tissues from NPC and non-NPC patients. BHRF1 transcripts were specifically detected in the NPC tumours (32 out of 44, 72.7%) rather than the non-NPC tissues (0 out of 25) by reverse transcription, polymerase chain reaction and Southern hybridization. Other EBV genes, such as the lytic gene BZLF1 and latent genes EBNA1 and LMP2A, were also investigated. BZLF1 transcripts also were found specifically in NPC tumours (33 out of 44, 75%). EBNA1 was expressed in 79.5% of NPC, and 28% of non- NPC, tissues and LMP2A was expressed in 70.5% of NPC, and 88% of non-NPC, tissues. BHRF1 protein was detected by immunohistochemistry in 4 metastatic NPC, of 36 NPC tissue sections available. The BHRF1 protein was distributed in both the nucleus and cytoplasm of the neoplastic epithelial cells. IgG antibody against the BHRF1 protein was detected in 6 of 17 (35. 3%) NPC plasma, but the protein and IgG were both absent from the non-NPC controls. BHRF1 DNA sequences were determined for 11 NPC and 3 non-NPC samples. No sequence was specific for the EBV isolates from NPC tissue. Amino acids 79 and 88 always appeared in the same form, however, for every tested isolate and both were valine or leucine. This particular characteristic was not present in the B95-8 strain or in the corresponding regions of homologues, Bcl-2 and Bcl-XL, and was regarded as unique to Oriental EBV strains.

Late-onset holocarboxylase synthetase deficiency with homologous R508W mutation.

Holocarboxylase synthetase (HCS) is responsible for the biotinylation of pyruvate carboxylase, propionyl coenzyme A (CoA) carboxylase, beta-methylcrotonoyl CoA carboxylase, and acetyl CoA carboxylase. We report on a patient with HCS deficiency resulting in a rare metabolic disease. The patient, a 2-year-old boy, presented with vomiting, consciousness disturbance, and dyspnea. Laboratory examinations showed hyperglycemia, hyperammonemia, lactic acidosis, and excretion of large amounts of beta-hydroxyisovalerate and beta-methylcrotonylglycine in the urine. After 10 days of treatment with biotin 5 mg.kg-1.day-1, the abnormal organic acids in his urine had almost completely disappeared. There were no subsequent attacks, and his growth and development remained normal during 1 year of follow-up. Nucleotide sequence analysis of the HCS cDNA of the patient revealed a homozygous 1809C-->T (R508W) mutation. The R508W mutation is found worldwide, and might be associated with higher residual HCS activity than other mutations. Late-onset HCS deficiency cannot be differentiated clinically from biotinidase deficiency. Prompt and correct diagnosis is important for these biotin-responsive disorders.

Alcoholic beverage preference and risks of alcohol-related medical consequences: a preliminary report from the National Longitudinal Alcohol Epidemiologic Survey.

In studying the alcohol-morbidity association, a substantial amount of attention and efforts has been focused on volume of alcohol intake. Considerably less is known about the differential health effects of beverage types. The present study used a most recent national household survey of the U.S. general population on drinking practices, alcohol use disorders, and their associated disabilities. The prevalence of a broad range of alcohol-related diseases was examined with respect to preferred beverage type, as well as consumption level. Our findings showed a reduced health risk associated with beer and wine drinking for a number of physical disorders, and a somewhat favorable cardiovascular effect of these two beverage types in relation to abstention. Among preferrers of beer, wine, and liquor, the results indicate that liquor preference is associated with elevated morbidity for several medical consequences. However, interpretation of results and the public health implications of these findings need to be taken cautiously, because sociodemographic and other behavioral characteristics were not considered in this preliminary report.

Antibody against the Epstein-Barr virus BHRF1 protein, a homologue of Bcl-2, in patients with nasopharyngeal carcinoma.

The Epstein-Barr virus (EBV) open reading frame BHRF1, a homologue of the oncogene bcl-2, was cloned from a patient with nasopharyngeal carcinoma (NPC) and overexpressed in Escherichia coli. The resulting recombinant BHRF1 fusion protein, with an apparent molecular weight of 35 KD, was used as antigen in an immunoblotting assay for IgG antibody in human sera. Anti-BHRF1 antibody was detected in 57 (61.3%) of 93 patients with NPC, 5 (5.7%) of 87 patients with nonmalignant diseases of the nasopharynx, and in 1 (1.3%) of 78 healthy blood donors. The positivity rate in these nonmalignant patients was 4.4 times that of the normal controls. Negative results were observed in four patients with infectious mononucleosis and patients with other cancers, including 4 with esophageal cancer, 11 with lung cancer, 10 with lymphoma, 13 with gastric carcinoma, 10 with cervical carcinoma, and 10 with other head and neck cancers. Antibody neutralizing EBV DNase and IgA antibody to viral capsid antigen (VCA) were assayed in parallel. The results showed that 7.5% of the NPC patients were negative for anti-DNase and anti-VCA antibodies and EBV infection could be detected by the anti-BHRF1 antibody alone. The demonstration of anti-BHRF1 antibody in most NPC sera strongly supports the hypothesis that the EBV BHRF1 protein is expressed in most NPC patients and its specific antibody can be a useful marker for the diagnosis of NPC.

Association of downregulation of cyclin D1 and of overexpression of cyclin E with p53 mutation, high tumor grade and poor prognosis in hepatocellular carcinoma.

BACKGROUND/AIMS: The major regulatory events leading to cell proliferation occur in the G1 phase of cell cycle, and the deranged expression of G1 cyclins is related to oncogenesis. In this study, we analyzed the aberrant expressions of cyclins D1 and E, and their role in hepatocellular carcinoma. METHODS: We examined paired hepatocellular carcinoma and liver RNAs taken from 71 patients who had been followed for more than 4 years after tumor resection, using reverse transcription-polymerase chain reaction supplemented with Northern blotting and immunohistochemistry. The genetic alterations of the p53 gene were also studied. RESULTS: Downregulation of cyclin D1 mRNA was detected in 29 hepatocellular carcinomas (40.8%), while overexpression was detected in only 4 hepatocellular carcinomas (5.6%). Downregulation of cyclin D1 was associated significantly with large hepatocellular carcinomas (p=0.0006) and poorly differentiated (grades III-IV) hepatocellular carcinoma (p=0.057), but not seen in any of 15 minute hepatocellular carcinomas (< or =2.5 cm in size). Cyclin E mRNA was overexpressed in 40 hepatocellular carcinomas, regardless of tumor size. Overexpression of cyclin E was significantly associated with poorly differentiated and invasive hepatocellular carcinoma (p=0.001 and p=0.015, respectively). Downregulation of cyclin D1 and overexpression of cyclin E were significantly associated with the p53 mutation (p=0.023 and p=0.005, respectively). Hepatocellular carcinomas expressing both downregulation of cyclin D1 and overexpression of cyclin E had the worst 4-year survival (p<0.03), and higher frequencies of the p53 mutation (p<0.001), large hepatocellular carcinoma (p<0.001), and invasive tumor (p<0.01). CONCLUSIONS: The deranged expressions of G1 cyclins correlate with the p53 mutation, tumor progression, and tumor biologic behavior of hepatocellular carcinoma. Overexpression of cyclin E occurs early, and downregulation of cyclin D1 late in hepatocellular carcinoma growth.

The relationship between drinking and heart disease morbidity in the United States: results from the National Health Interview Survey.

This study, based on data drawn from the responses of 18,323 males and 25,440 females to the 1988 National Health Interview Survey, a nationally representative, multistage probability sample of the United States, attempts to define more precisely the level of drinking at which the relationship between heart disease and alcohol consumption is a protective one. Its attempt at precision derives from (1) using drinking categories that represent various points within the range of moderate drinking (1-6 drinks) defined in the literature as protective; (2) adjusting for underreporting that commonly occurs in population surveys by using consumption at time of heaviest drinking; and (3) controlling for age, body mass, smoking, former drinker, and former smoker status, duration of drinking, and sociodemographic factors. It also examines whether the relationship derived from these levels conforms to the U-shaped curve that demonstrates the protective effect of moderate drinking when abstainers are not used as the reference group. Relative to infrequent drinkers (less than 1 drink per day), men report more heart disease at the level of more than five drinks per day. However, black men also report more heart disease, relative to infrequent drinkers, at the greater than two drinks per day level; and women report more heart disease at the level of more than two drinks per day at the time of their heaviest drinking. Former drinkers of both genders, considered as an independent variable in the regression analysis, were more likely to report having heart disease. Abstainers, light drinkers, and infrequent drinkers were not significantly different in their reports of heart disease. Our results are consistent with studies that suggest protection from heart disease occurs only at lower levels of drinking.

Medical consequences of alcohol consumption--United States, 1992.

There is plenty of evidence in the alcohol literature that chronic excessive use of alcohol poses a threat to every organ system in the body. At the same time, there is a growing consensus that drinking in moderation protects against cardiovascular disease. This study was based on the most recent national household survey of the United States general population on drinking practices, alcohol use disorders, and their associated disabilities. The prevalence of major alcohol-related diseases were examined across different categories of drinking status. Excess morbidity caused by heavy intake of alcohol was also studied. Results were generally in agreement with the popular belief that light or moderate drinking is beneficial relative to abstention, particularly that moderate alcohol consumption confers a beneficial cardiovascular effect. Our findings also pointed toward the injurious effect of heavy alcohol use. However, results on benefits of drinking must be interpreted with caution.

Metabolic function and liver histopathology in Reye-like illnesses.

Forty children with Reye syndrome (RS) or Reye-like illnesses were investigated to elucidate the underlying aetiologies. Extensive biochemical studies including patterns of organic acids and amino acids, liver histopathology, and, if available, a DNA approach were performed. In addition to classical RS (n = 10), the causes of Reye-like conditions included hereditary organic acidaemias (n = 13), urea cycle defects (n = 4), mitochondrial disorders (n = 3), fulminant hepatitis (n = 2), tyrosinaemia (n = 1), valproate-associated hepatotoxicity (n = 1), and other non-specific generalized organic acid disorders (n = 6). It is important to collect specimens when encephalopathy with liver dysfunction of unknown causes is noted. When the underlying inherited metabolic disorders are confirmed, the prevention of the recurrence by adequate diet control and medications, and genetic counselling become possible.

Subgroup variation in U.S. drinking patterns: results of the 1992 national longitudinal alcohol epidemiologic study.

Data from the 1992 National Longitudinal Alcohol Epidemiologic Study (NLAES) revealed that 44% of U.S. adults 18 years of age and older were current drinkers who had consumed at least 12 drinks in the year preceding the interview. Twenty-two percent were former drinkers, and 34% were lifetime abstainers. These figures represent an 8% decrease in the prevalence of current drinking relative to 1988. The proportion of current drinkers decreased with age, was higher for men than women, increased with education and income, was lower than average among Black and Hispanic adults, was highest among never-married adults and lowest among those who were widowed, was lower in the South than in other regions, and was lower in rural than urban areas. The probabilities of ever having consumed five or more (5+) drinks or having been intoxicated in the past year revealed similar patterns to those already noted, but the probabilities of heavy drinking or intoxication on a weekly or more frequent basis showed no variation by race or ethnicity. Average daily consumption of more than 1 ounce of ethanol differed from the preceding measure of heavy drinking in its variation across population subgroups, declining less sharply with age and exhibiting a U-shaped pattern with respect to income. Examination of the prevalence of heavy drinking among current drinkers rather than within the total population revealed several differences, the most striking reversal being that the probability of heavy drinking decreased with education and income. Multiple logistic regression models predicting the various drinking outcomes indicated that most of the differentials held true after adjusting for intercorrelation among the sociodemographic variables.

An examination of the alcohol consumption and peptic ulcer association--results of a national survey.

Excessive alcohol consumption causes damages to the stomach or duodenum by impairing the integrity of the mucosal barrier. The aim of this study was to estimate the association between alcohol consumption and peptic ulcer, utilizing a large representative sample of the U.S. population, while controlling for cigarette smoking and major sociodemographic variables. Results indicated that alcohol consumption only minimally increased the odds of peptic ulcer. Thus, this study offers little support for the association between ethanol intake and peptic ulcer.

Bradykinin-stimulated phosphoinositide metabolism in cultured canine tracheal smooth muscle cells.

1. Stimulation of bradykinin (BK) receptors coupled to phosphoinositide (PI) hydrolysis was investigated in canine cultured tracheal smooth muscle cells (TSMCs). BK, kallidin, and des-Arg9-BK, stimulated [3H]-inositol phosphates (IPs) accumulation in a dose-dependent manner with half-maximal responses (EC50) at 20 +/- 5, 13 +/- 4, and 2.3 +/- 0.7 nM, (n = 5), respectively. 2. D-Arg[Hyp3, D-Phe7]-BK and D-Arg[Hyp3, Thi5,8, D-Phe7]-BK, B2 receptor antagonists, were equipotent in blocking the BK-induced IPs accumulation with pKB = 7.1 and 7.3, respectively. 3. Short-term exposure of TSMCs to phorbol 12-myristate 13-acetate (PMA, 1 microM) attenuated BK-stimulated IPs accumulation. The concentrations of PMA that gave half-maximal and maximal inhibition of BK-induced IPs accumulation were 15 +/- 4 nM and 1 microM, n = 3, respectively. The inhibitory effect of PMA on BK-induced response was reversed by staurosporine, a protein kinase C (PKC) inhibitor, suggesting that the inhibitory effect of PMA was mediated through the activation of PKC. 4. Prolonged incubation of TSMCs with PMA for 24 h, resulted in a recovery of receptor responsiveness which may be due to down-regulation of PKC. The inactive phorbol ester, 4 alpha-phorbol 12, 13-didecanoate at 1 microM, did not inhibit this response. 5. The site of this inhibition was further investigated by examining the effect of PMA on AlF(4-)-induced IPs accumulation in canine TSMCs. AlF(4-)-stimulated IPs accumulation was inhibited by PMA treatment, suggesting that the G protein(s) can be directly activated by AlF4-, which is uncoupled from phospholipase C by PMA treatment. 6. Incubation of TSMCs in the absence of external Ca2+ or upon removal of Ca2+ by addition of EGTA, caused a decrease in IPs accumulation without changing the basal levels. Addition of Ca2+ (3-620 nM) to digitonin-permeabilized TSMCs stimulated IPs accumulation was obtained by inclusion of either guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) or BK. The combination of GTP gamma S and BK caused an additive effect on IPs accumulation.7. Pretreatment of TSMCs with cholera toxin enhanced BK-stimulated IPs accumulation, whereas there was no effect with pertussis toxin.8. These data suggest that BK-stimulated PI metabolism is mediated by the activation of BK B2 receptors coupling to a G protein which is not blocked by cholera toxin or pertussis toxin treatment and dependent on external Ca2+. The transduction mechanism of BK coupled to PI hydrolysis is sensitive to feedback regulation by PKC.

Sex differences in morbidity among respondents classified as alcohol abusers and/or dependent: results of a national survey.

To date, none of the studies on gender differences in physical morbidity have focused on persons classified as DSM-III-R alcohol abusers and/or dependent in the general population. This Data Note presents data from a nationally representative survey on drinking practices and related problems for the purpose of examining gender differences in physical morbidity among respondents receiving these diagnoses. Results indicated that for certain major sociodemographic subgroups of the population, gender differences in morbidity were significant. The female-to-male odds ratios of these subgroups generally varied within the range of 1.5 and 2.0, reflecting about two times greater odds of experiencing morbid conditions for females when compared to males.

A study of the gender differences in morbidity among individuals diagnosed with alcohol abuse and/or dependence.

The majority of studies on medical consequences of excessive alcohol consumption have been carried out with male subjects, mostly from clinical or hospitalized samples. The purpose of this report was to study differences in morbidity outcomes of men and women among respondents diagnosed with alcohol abuse and/or dependence. Utilizing data from the 1988 National Health Interview Survey, this study compared several indicators of physical morbidity among male and female respondents meeting the criteria for Diagnostic and Statistical Manual of Mental Disorders (DSM-III-R; American Psychiatric Association, 1987) alcohol abuse and/or dependence. The results revealed complex gender differences, not all of which can be explained by differences in sociodemographic characteristics or drinking practices. Moreover, the results indicated that it is inadequate to generalize results based on morbidity data of men with alcohol abuse and/or dependence to their female counterparts or female drinkers. Implications of these findings are discussed.

Muscarinic regulation of cytosolic free calcium in canine tracheal smooth muscle cells: Ca2+ requirement for phospholipase C activation.

1. The relationship between muscarinic receptor-mediated phosphatidylinositol 4,5-bisphosphate (PIP2) breakdown and the increase of intracellular Ca2+ ([Ca2+])i has been examined in canine cultured tracheal smooth muscle cells (TSMCs). 2. Addition of acetylcholine (ACh) and carbachol led to a 2-3 fold increase in [Ca2+]i over the resting level as determined by fura-2, with half-maximal stimulation (EC50) obtained at concentrations of 97 and 340 nM, respectively. Addition of the partial agonist, bethanechol, showed a smaller increase in PIP2 turnover and [Ca2+]i than did ACh or carbachol. 3. Addition of ACh or carbachol to TSMCs that had been prelabelled with [3H]-inositol led to the rapid (5-15 s) release of inositol mono, bis and trisphosphates IP1, IP2 and IP3. The time course of IP3 accumulation is correlated with the time course of the peak rise in [Ca2+]i. 4. Inclusion of EGTA lowered the resting [Ca2+]i and markedly reduced the extent of the agonist-induced rise in [Ca2+]i. When assayed under conditions similar to those used for the [Ca2+]i measurements, EGTA reduced the muscarinic agonist-stimulated inositol phosphates (IPs) accumulation. Conversely, ionomycin could stimulate IPs accumulation and elevate [Ca2+]i. The addition of Ca2+ (2.7-617 nM) to digitonin-permeabilized TSMCs directly stimulated IPs accumulation. 5. Both Ca2+ and guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) stimulated the formation of IPs in digitonin-permeabilized TSMCs prelabelled with [3H]-inositol. A further calcium-dependent increase in IPs accumulation was obtained by inclusion of either GTP gamma S or carbachol. The combined presence of carbachol and GTP gamma S elicited a synergistic effect on IPs accumulation, with half-maximal stimulation observed at approximately 8 nM free Ca2+.6. These results indicate that (i) the magnitude of the initial rise in [Ca2+], is directly related to the production of IPs and (ii) the phospholipase C-mediated PIP2 breakdown in TSMCs is sensitive to regulation by physiologically relevant concentrations of free Ca2+ ([Ca2+]f).

Fragile-X mental retardation--a combination of cytogenetic and molecular approaches, with greater emphasis on DNA analysis.

Fragile X syndrome is one of the most frequent causes of hereditary mental retardation. In the past, its diagnosis depended primarily on cytogenetic demonstration of chromosome fragile site Xq27.3. Recently, the gene FMR-1 has been found responsible for this disease. Here a combined method was used to study fragile X syndrome. A fragment (pP1fr) of DNA was subcloned from pE5.1 by polymerase chain reaction. With this probe, DNA samples from two cytogenetically proved families were analyzed by restriction fragment length polymorphisms. It was demonstrated that EcoRI polymorphism was an easy and accurate method for diagnosis of the fragile X syndrome. To study methylation status of patients, another methylation-sensitive enzyme, BssHII, could be used together with EcoRI. The PstI polymorphism of one family was also studied and showed one kb fragment as normal, and detected more precise changes in length. Prominent mosaicism necessary was characteristic in PstI polymorphism. The DNA diagnosis of fragile X syndrome was a reliable method.

Primary culture of canine tracheal smooth muscle cells in serum-free medium: effects of insulin-like growth factor I and insulin.

The effects of growth factors on cell growth and muscarinic receptor (mAChR) expression of canine tracheal smooth muscle cells (TSMCs) were observed under serum-free medium supplemented with 0.1% BSA. In the presence of 0.1% BSA, TSMCs withdraw from cell cycle as compared with 10% FBS and allow to determine the effects of growth factors on mAChR expression. The individual components of growth factors (IGF-I, insulin, and aFGF) at the concentration used are not sufficient to stimulate growth of TSMCs in the primary culture with 0.1% BSA. IGF-I (10 ng/ml) and insulin (1 microgram/ml), alone or in combination, could stimulate the expression of mAChRs of cultured TSMCs. Heparin could inhibit these stimulatory effects of mAChR expression. The stimulatory effects of IGF-I and insulin on mAChR expression were mediated through their own receptors since these effects were reversed by pretreatment of TSMCs with antibodies of the respective growth factor receptors. The pharmacological response of functional mAChRs, determined as accumulation of inositol phosphates induced by carbachol, is greater in the medium containing IGF-I and insulin than that cultured in 0.1% BSA. These results firmly establish that IGF-I and insulin could stimulate the expression of mAChRs in TSMCs under serum-free culture condition.