[The value of DISCO and MUSE-DWI combined with prostate specific antigen density in the diagnosis and risk stratification of prostate cancer].

Objective: To explore the value of differential subsampling with cartesian ordering (DISCO) and multiplexed sensitivity-encoding diffusion weighted-imaging (MUSE-DWI) combined with prostate specific antigen density (PSAD) in the diagnosis and risk stratification of prostate cancer (PCa). Methods: The data of 183 patients [aged from 48 to 86 (68±8) years] with prostate diseases in the General Hospital of Ningxia Medical University from July 2020 to August 2021 were retrospectively collected. Those patients were divided into non-PCa group (n=115) and PCa group (n=68) based on the disease condition. According to the risk degree, PCa group was subdivided into low risk PCa group (n=14) and medium-to-high risk PCa group (n=54). The differences of volume transfer constant (Ktrans), rate constant (Kep), extracellular volume fraction (Ve), apparent diffusion coefficient (ADC) and PSAD between groups were analyzed. Receiver operating characteristic (ROC) curves analysis were conducted for evaluating the diagnostic efficacy of quantitative parameters and PSAD in distinguishing non-PCa and PCa, low-risk PCa and medium-high risk PCa. Multivariate logistic regression model was used for screening out the predictors, which was statistically significant differences between non-PCa group and PCa group, for PCa prediction. Results: Ktrans, Kep, Ve and PSAD of PCa group all were higher than those of non-PCa group, and ADC value was lower than that of non-PCa group, and the differences all were statistically significant (all P<0.001). Ktrans, Kep and PSAD of medium-to-high risk PCa group all were higher than those of low risk PCa group, and ADC value was lower than that of low risk PCa group, and the differences were all statistically significant (all P<0.001). When distinguishing non-PCa from PCa, the area under ROC curve (AUC) of the combined model (Ktrans+Kep+Ve+ADC+PSAD) was higher than that of any single index [0.958 (95%CI: 0.918-0.982) vs 0.881 (95%CI: 0.825-0.924), 0.836 (95%CI: 0.775-0.887), 0.672 (95%CI: 0.599-0.740), 0.940(95%CI: 0.895-0.969), 0.816(95%CI:0.752-0.869), all P<0.05]. When distinguishing low-risk PCa and medium-to-high risk PCa, the AUC of the combined model (Ktrans+Kep+ADC+PSAD) were higher than those of Ktrans, Kep and PSAD[0.933 (95%CI: 0.845-0.979) vs 0.846 (95%CI:0.738-0.922), 0.782 (95%CI:0.665-0.873), 0.84 8(95%CI: 0.740-0.923), all P<0.05]. The multivariate logistic regression analysis showed that Ktrans (OR=1.005, 95%CI:1.001-1.010) and ADC values (OR=0.992, 95%CI:0.989-0.995) were predictors of PCa (P<0.05). Conclusions: DISCO and MUSE-DWI combined with PSAD can distinguish benign and malignant prostate lesions. Ktrans and ADC values were predictors of PCa; Ktrans, Kep, ADC values and PSAD are helpful in predicting the biological behavior of PCa.

[The value of relaxation time quantitative technique from synthetic magnetic resonance imaging in the diagnosis and invasion assessment of prostate cancer].

Objective: To investigate the application value of relaxation time quantitative technique from synthetic magnetic resonance imaging (MRI) in the diagnosis and invasion assessment of prostate cancer. Methods: A total of 119 patients with prostate diseases [122 regions of interest(ROI)] who underwent routine MRI scan and magnetic resonance image compilation (MAGiC) sequence of prostate from March 2020 to March 2021 in General Hospital of Ningxia Medical University were retrospectively collected, they were divided into prostate cancer group(58 cases, 61 ROI) and non-prostate cancer group(61 cases, 61 ROI) according to the pathological results. In the prostate cancer group, those patients with an age of 48 to 85(69.8±5.9) years, and further divided into two subgroups according to the location of occurrence: peripheral zone cancer group (43 cases, 45 ROI) and transitional zone cancer group (15 cases, 16 ROI). The non-prostate cancer group consisted of patients with benign prostatic hyperplasia or complicated with chronic prostatitis, with an age of 41 to 81(68.6±7.0) years, and they were further divided into two subgroups according to the location of occurrence: non-cancerous peripheral zone group (45 cases, 45 ROI) and transitional zone benign prostatic hyperplasia group(16 cases, 16 ROI). Prostate cancer lesions were classified as low risk (Gleason score ≤6) or intermediate/high risk (Gleason score ≥7). After the post-processing of MAGiC images, T1, T2 and proton density(PD) values of prostate cancer group and non-prostate cancer group were obtained. At the same time, relevant software were used for image post-processing to generate apparent diffusion coefficient (ADC) value, the data between the two groups were analyzed by the Independent sample t-test or Mann-Whitney U-test, and the diagnostic effectiveness of each quantitative parameter in diagnosing prostate cancer and discriminating low risk prostate cancer from intermediate/high risk prostate cancer was analyzed by using receiver operating characteristic curve (ROC) analysis, the correlation between each quantitative parameter and Gleason score were assessed by Spearman correlation analysis. Results: The T1 value and T2 value of the peripheral zone cancer group were lower than those in non-cancerous peripheral zone group [1 201.3 (1 103.5, 1 298.2) ms vs 2 274.0 (1 620.9, 2 776.5) ms; 78.0 (74.0, 83.8) ms vs (160.6±54.9) ms] (all P<0.001), there was no statistically significant in PD value between the two groups (P>0.05). The T1 value and T2 value of the transitional zone cancer group were lower than those in transitional zone benign prostatic hyperplasia group [1 073.3 (1 003.9, 1 164.9) ms vs 1 340.8 (1 208.5, 1 502.8) ms; 76.9 (74.8, 82.8) ms vs 95.1(82.8, 103.4) ms] (all P<0.001), there was no statistically significant in PD value between the two groups (P>0.05). The area under the curve (AUC) of T2 value was similar with the ADC value in discriminating peripheral zone cancer group from non-cancerous peripheral zone group(0.963 vs 0.991, P=0.105), while in discriminating transitional zone cancer group from transitional zone benign prostatic hyperplasia group, the AUC of T2 value、T1 value and ADC value were similar(0.867, 0.930 vs 0.938, all P>0.05). ADC value, T2 value all were negatively correlated with Gleason score (r=-0.747,-0.453, all P<0.001). T2 value and ADC value demonstrated equivalent diagnostic performance in discriminating low risk from intermediate/high risk prostate cancer, and there were no statistically significant (AUC: 0.787 vs 0.943, P=0.069). Conclusions: Quantitative relaxation time T1 and T2 values derived from synthetic MRI can discriminate prostate cancer from other benign pathologies, and T2 value have the equivalent diagnostic performance compared to ADC value. Synthetic MRI has high clinical application value, and T2 value can distinguish low risk prostate cancer from intermediate/high risk prostate cancer.

[Clinicopathological analysis of 11 cases of hepatic amyloidosis].

Objective: Hepatic amyloidosis is a metabolic disease with a low incidence rate. However, because of its insidious onset, the rate of misdiagnosis is high, and it usually progresses to a late stage when it is diagnosed. This article analyzes the clinical features of hepatic amyloidosis by combining clinical pathology in order to improve the clinical diagnosis rate. Methods: Clinical and pathological data of 11 cases of hepatic amyloidosis diagnosed at the China-Japan Friendship Hospital from 2003 to 2017 were summarized and analyzed retrospectively. Results: The clinical manifestations of 11 cases mainly included abdominal discomfort (4/11), hepatomegaly (7/11), splenomegaly (5/11), fatigue (6/11), etc. Biochemical test results showed that most patients' alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, γ-glutamyl transferase, total bilirubin, direct bilirubin, and total bile acids, accompanied by hypoalbuminemia were elevated, while some patients' 24-h urinary protein, creatinine, and blood urea nitrogen were elevated. Conclusion: All patients had slightly elevated aspartate transaminase levels (within 5 times the upper limit of normal), and 72% had slightly elevated alanine transaminase. Alkaline phosphatase and γ-glutamyl transferase levels were significantly raised in all cases, with the highest result for γ-glutamyl transferase being 51 times the upper limit of normal. Damage to the hepatocytes has an effect on the biliary system as well, leading to symptoms such as portal hypertension and hypoalbuminemia [(0.54~0.63) × upper limit of normal value, 9/11]. Amyloid deposits within the artery wall (54.5% of patients) and portal vein (36.4% of patients) were also indicative of vascular injury. A liver biopsy should be recommended for patients with unexplained elevated transaminases, bile duct enzymes, and portal hypertension in order to establish a definitive diagnosis.

Oxymatrine therapy inhibited epidermal cell proliferation and apoptosis in severe plaque psoriasis.

BACKGROUND: Psoriasis is a chronic skin disorder that manifests as epidermal keratinocyte hyperplasia. OBJECTIVES: We examined the effect of oxymatrine treatment on cell proliferation and apoptosis in skin lesions of psoriasis. PATIENTS AND METHODS: Patients with severe plaque psoriasis were treated with oxymatrine or with acitretin. The skin lesions were stained with proliferating cell nuclear antigen (PCNA), Ki-67 and Bcl-2, as well as examined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL). We performed correlations of the Psoriasis Area and Severity Index (PASI) and the proliferation and apoptosis index. RESULTS: Oxymatrine significantly reduced the psoriasis lesions as demonstrated by the reduced PASI score after treatment [6·91; 95% confidence interval (CI) 5·00-8·81, P < 0·001]. In the oxymatrine group, the mitotic index was 26·15 (95% CI 24·80-27·49) before oxymatrine treatment, decreasing to 14·52 (95% CI 13·82-15·25; P < 0·001) after treatment, but remained higher than the normal group (6·24; 95% CI 5·87-6·61, P < 0·001). Oxymatrine also inhibited the proliferation of epidermal cells in the skin lesion as indicated by the reduced proliferation index after treatment (P < 0·01). In addition, oxymatrine treatment reduced cellular apoptosis as shown by increased Bcl-2 expression and a decrease in TUNEL-positive cells. The PASI score was positively correlated with mitotic index, proliferation index and apoptotic index (TUNEL), but negatively correlated with Bcl-2 expression. CONCLUSIONS: Oxymatrine treatment reduced proliferation but inhibited apoptosis of cells in the skin lesion. The balance between cell proliferation and turnover may contribute to the significant alleviation of psoriasis by oxymatrine. What's already known about this topic? Psoriasis manifests as epidermal keratinocyte hyperplasia with proliferation, keratinocyte maturation and turnover rates. Current drugs for psoriasis may inhibit cell proliferation but could not adjust the balance of cell division, differentiation and apoptosis. What does this study add? We studied the efficacy of oxymatrine in the treatment of psoriasis and analysed the correlation of skin lesions, proliferation and apoptosis index before and after oxymatrine treatment. What is the translational message? Our study has demonstrated that oxymatrine is effective in the treatment of severe plaque psoriasis. It has comparable efficacy with acitretin. Because acitretin treatment was sometimes associated with metabolic abnormalities, our study suggests oxymatrine therapy as an alternative treatment for psoriasis in the context of acitretin allergy or adverse reactions.

[Value and related factors of preoperative diagnosis of extramural vascular invasion of rectal cancer by 3.0T magnetic resonance imaging].

Objective: To evaluate the value of preoperative diagnosis of extramural vascular invasion (EMVI) of rectal cancer with 3.0T high-resolution magnetic resonance imaging (MRI) and the MRI-related factors of EMVI in rectal cancer. Methods: The clinical and imaging data of 40 patients with rectal cancer were retrospectively analyzed. The postoperative pathological diagnosis was used as the gold standard to evaluate the diagnostic efficacy of preoperative diagnosis of EMVI of rectal cancer by high-resolution MRI, and to analyze the relationship between the EMVI and clinical and MRI features. Results: Of the 40 patients, 19 cases were diagnosed as positive EMVI and 21 were negative by MRI. Pathological diagnosis of EMVI was positive in 10 cases and negative in 30 cases. The sensitivity, specificity and accuracy of MRI in the diagnosis of EMVI were 100%, 70.0% and 77.5%, respectively. Preoperative MRI and postoperative pathology were moderately consistent in the diagnosis of EMVI in rectal cancer (Kappa=0.538, P<0.001). Pathological EMVI positivity were related to tumor size under MRI examination (P=0.028), degree of differentiation (P<0.001), depth of invasion (P=0.002), lymph node metastasis (P=0.001), liver metastasis (P=0.011), tumor apparent diffusion coefficient (ADC) value (P=0.010) and exponential apparent diffusion coefficient (eADC) value (P=0.003). It also related to extramural nerve invasion by pathological examination (P=0.005). Conclusion: According to the EMVI imaging score of rectal cancer, preoperative MRI has a high value in the diagnosis of EMVI of rectal cancer.

[Combined anluohuaxianwan and entecavir treatment significantly improve the improvement rate of liver fibrosis in patients with chronic hepatitis B virus infection].

Objective: To explore the improvement rate of liver fibrosis in patients with chronic hepatitis B virus infection who received entecavir alone or in combination with anluohuaxianwan for 78 weeks. Methods: Patients with chronic HBV infection were randomly treated with entecavir alone or in combination with anluohuaxian for 78 weeks. Ishak fibrosis score was used for blind interpretation of liver biopsy specimens. The improvement in liver fibrosis condition before and after the treatment was compared. Student's t test and non-parametric test (Mann-Whitney U-Test and Kruskal-Wallis test) were used to analyze the measurement data. The categorical variables were analyzed by Chi-square test method and Spearman's rank correlation coefficient was used to test bivariate associations. Results: Liver fibrosis improvement rate after 78 weeks of treatment was 36.53% (80/219) and the progression rate was 23.29% (51/219). The improvement of liver fibrosis was associated to the degree of baseline fibrosis and treatment methods (P < 0.05). The improvement rate of hepatic fibrosis in patients treated with anluohuaxianwan combined with entecavir at baseline F < 3 (54.74%, 52/95) was significantly higher than that in patients treated only with entecavir (33.33%, 16/48), P = 0.016 and the progression rate of hepatic fibrosis (13.68%, 13/95) was lower than that in patients treated alone (18.75%, 9/48), P = 0.466. In patients with baseline F < 3, the proportion of patients with improved and stable liver fibrosis in the combined treatment group (68.1%, 32/47) was higher than that in the treatment group alone (51.7%, 15/29). Conclusion: Combined anluohuaxianwan and entecavir treatment can significantly improve the improvement rate of liver fibrosis in patients with chronic hepatitis B virus infection. Furthermore, it has the tendency to improve the stability rate and reduce the rate of progression of liver fibrosis.

Gender Concept, Work Pressure, and Work-Family Conflict.

Based on the data of the 2015 China General Social Survey (CGSS), this article empirically analyzed the influence of gender concept, work pressure, and work flexibility on work-family conflict (work interfering family (WIF) and family interfering work (FIW)) from three perspectives (gender, age, and urban and rural areas in China) and tested its significance. The empirical results show that individuals holding the concept of gender inequality produced lower WIF and FIW, which only exists between sexual relations, older working people, and urban and rural areas. Multicultural exchange and integration only made it easier for working people under the age of 30 to accept the concept of gender equality, but it increased their WIF and FIW. Second, with the development of the economy and society of China, the work pressure of workers is the most important factor causing WIF and FIW. Lastly, in order to cope with the pressure of employment and the cost of living, it is difficult to ease the conflict between work and family.

Carbachol-induced reverse transformation of Chinese hamster ovary cells transfected with and expressing the m5 muscarinic acetylcholine receptor.

Reverse transformation was induced in Chinese hamster ovary (CHO) cells transfected with and stably expressing the m5 subtype of the muscarinic acetylcholine receptor when stimulated with the muscarinic agonist, carbachol. Atropine, a muscarinic antagonist, blocked the carbachol-stimulated reverse transformation. CHO cells not transfected with the muscarinic receptor did not change with added carbachol. PMA induced reverse transformation without increasing cAMP accumulation in CHO cells. Carbachol, prostaglandin E2, and cholecystokinin increased cAMP accumulation but only carbachol caused reverse transformation. Carbachol-stimulated cAMP accumulation occurred at a higher concentration (EC50 10 microM) than did carbachol-stimulated reverse transformation (EC50 63 nM). Muscarinic m5 acetylcholine receptor transfected into CHO cells can induce reverse transformation which may be independent of cAMP.

Independent induction of morphological transformation of CHO cells by receptor-activated cyclic AMP synthesis or by receptor-operated calcium influx.

Morphological transformation of Chinese hamster ovary (CHO) cells can be induced by exogenous addition of cyclic AMP (cAMP) or through the stimulation of G protein-coupled receptors ectopically expressed in these cells. The morphological transformation has been shown to represent a phenotypic suppression of CHO cell tumorigenic potential. Studies were undertaken to determine which receptor-activated signal transduction pathway initiates the progression from a tumorigenic to a non-tumorigenic phenotype. Stimulation of CHO cells expressing the dopamine D1 receptor (CHOD1) with a D1 selective agonist, SKF38393, resulted in an increase in cAMP accumulation which correlated with morphologic transformation. SKF38393 had no effect on intracellular calcium levels, arguing against a requirement for phospholipase C or calcium mobilization in the D1-stimulated morphology change. In contrast, stimulation of muscarinic m5 (CHOm5) or vasopressin V1a (CHOV1a) receptors expressed in CHO cells with carbachol or arginine vasopressin (AVP), respectively, did not result in an increase in intracellular calcium and a morphology change. The time course of carbachol-stimulated calcium influx correlated with the time course of morphological transformation, but not with carbachol-stimulated cAMP or inositol, 1,4,5-trisphosphate (IP3) accumulation. Furthermore, no increase in cAMP accumulation was observed in AVP-stimulated CHOV1a cells, suggesting a cAMP-independent stimulation of the transformation process. Carbachol-stimulated CHO cells expressing the m2 muscarinic receptor (CHOm2) failed to undergo a morphological transformation, yet released IP3. Therefore, phospholipase C-mediated signal transduction is not sufficient for the morphological transformation of CHO cells. It appears that receptor-stimulated morphologic transformation of CHO cells can be induced via two independent signaling pathways, mediated by adenylate cyclase or receptor-operated calcium channels.

Muscarinic acetylcholine receptor subtypes associated with release of Alzheimer amyloid precursor derivatives activate multiple signal transduction pathways.

Five subtypes of muscarinic acetylcholine receptors have been identified and designated m1-m5. The m1 and m3 receptors have recently been shown to stimulate APP processing. The m1 and m3 receptors couple to a variety of signal transduction pathways in both tissue slices and a variety of cell lines endogenously expressing either or both subtypes. In contrast, the m2 and m4 receptors have been primarily associated with inhibition of adenylate cyclase. We have transfected all five subtypes of muscarinic receptors into a variety of mammalian cell lines in order to investigate the signaling associated with single receptor subtypes. The m1, m3, or m5 receptors stimulate phospholipase A2, C, and D, adenylate cyclase, receptor-operated calcium channels, and tyrosine kinase activity simultaneously. The m2 or m4 receptor inhibits cAMP accumulation and augments a previously stimulated release of arachidonic acid and calcium influx.

Muscarinic receptors mediate the release of arachidonic acid from spinal cord and hippocampal neurons in primary culture.

Muscarinic receptors are involved in CNS neurotransmissions and have been shown to transduce their message by modulating cAMP, calcium, inositol phosphates, and more recently, by liberating arachidonic acid via phospholipase A1. We have previously shown that the alpha 1-adrenergic and 5-HT2 serotonergic neurotransmitter receptors cause the release of arachidonic acid from spinal cord and hippocampal neurons, respectively, in primary culture. In this study, we demonstrated a muscarinic receptor-mediated release of arachidonic acid in these two neural segments which occurred independent of phosphatidylinositol-specific phospholipase C. This release of arachidonic acid was neuronal (not glial) in origin and exhibited M1 muscarinic receptor pharmacology.

[Experimental study on effect of sanmiao mixture capsules on prostate hyperplasia in mice and rats].

OBJECTIVE: To observe the effect of Sanmiao Mixture Capsules(SMC) on prostate hyperplasia in mice and rats. METHOD: The model of prostate hyperplasia was made by injecting testosterone propionate in to male mice(5 g.kg-1.d-1, 21 d) and rats(3 g.kg-1.d-1, 14 d). The treated group was administered SMC(mice: 36.3 g.kg-1 and 18.2 g.kg-1; rats: 25.2 g.kg-1 and 12.6 g.kg-1), the normal control group 1.9 g.kg-1, and the model control group NS. hours after the last administration serum tests were carried out on E2, AKP and Zn2+. Then the animals were killed, prostates taken out and weighed, index of prostate was calculated and pathological examination performed. RESULT: In the SMC treated group, the prostate weight and index were lowered(P < 0.01) the mean activation of E2 was raised, and the mean concentration of AKP and Zn2+ was inhibited (P < 0.01). CONCLUSION: SMC are helpful in checking prostate hyperplasia in mice and rats, the mechanism being probably related to the raising of activation of E2 as well as to the inhibition of concentration of AKP and Zn2+.

Cryptococcal meningitis in an immunocompetent adolescent.

Cryptococcus neoformans is a rare cause of meningitis especially in immunocompetent children. Cryptococcal meningitis in a healthy Chinese adolescent is reported. She responded well to treatment with intravenous amphotericin and oral flucytosine.

Photoperiod and testosterone interact to drive seasonal changes in kisspeptin expression in Siberian hamsters (Phodopus sungorus).

Kisspeptin, a neuropeptide product of the KiSS-1 gene, has recently been implicated in the regulation of seasonal breeding in a number of species, including Siberian hamsters. In this species, kisspeptin expression is reduced in the anteroventral periventricular nucleus (AVPV) following exposure to inhibitory day lengths, and exogenous kisspeptin activates the reproductive neuroendocrine axis of reproductively quiescent animals. Because sex steroids can impact kisspeptin expression, it is unclear whether changes in kisspeptin occur in direct response to photoperiodic cues or secondarily in response to changes in sex steroid concentrations resulting from the transition to reproductive quiescence. The present study aimed to assess the relative contributions of photoperiod and testosterone in regulating kisspeptin expression in Siberian hamsters. Animals housed in long or short day lengths for 8 weeks were either castrated or received sham surgeries. Half of the hamsters in each photoperiod were given testosterone to mimic long-day sex steroid concentrations. The results obtained indicate that kisspeptin neurones in the AVPV and arcuate nuclei were influenced by both photoperiod and testosterone. In the AVPV, removal of testosterone or exposure to inhibitory day lengths led to a marked reduction in kisspeptin-immunoreactive cells, and testosterone treatment increased cell numbers across conditions. Importantly, long-day castrates exhibited significantly more kisspeptin cells than short-day castrates or intact short-day animals with empty capsules, suggesting the influences of photoperiod, independent of gonadal steroids. In general, the opposite pattern emerged for the arcuate nuclei. Collectively, these data suggest a role for both gonadal-dependent and independent (i.e. photoperiodic) mechanisms regulating seasonal changes in kisspeptin expression in Siberian hamsters.

Phorbol esters and diacylglycerols amplify bradykinin-stimulated prostaglandin synthesis in Swiss 3T3 fibroblasts. Possible independence from protein kinase C.

When Swiss 3T3 fibroblasts were incubated with bradykinin, prostaglandin E2 (PGE2) synthesis was stimulated. Phorbol esters or the diacylglycerol analog 1-oleoyl-2-acetylglycerol (OAG), by themselves, did not acutely stimulate PGE2 synthesis. However, when cells were preincubated with phorbol esters or OAG, bradykinin-stimulated PGE2 synthesis was potentiated markedly. When phorbol esters and OAG were added together, bradykinin-stimulated PGE2 synthesis was potentiated in an additive manner. When cells were preincubated for 48 h with phorbol esters, then bradykinin added, amplification of bradykinin-stimulated PGE2 synthesis by phorbol ester or OAG was still apparent, even though prolonged pretreatment with phorbol esters abolished protein kinase C (Ca2+/phospholipid-dependent enzyme) activity in cell-free preparations. Further, the protein kinase C antagonist, H-7, only slightly inhibited phorbol ester or OAG amplification of bradykinin-stimulated PGE2 synthesis. The possibility is raised that diacylglycerol, formed in response to many receptors, may serve as a transducer of receptor-receptor interactions. Since desensitization or inhibition of protein kinase C only partially reduced the amplification of bradykinin-stimulated PGE2 synthesis by phorbol esters or OAG, the possibility is raised that diacylglycerol mimetics may have actions in addition to activation of protein kinase C.

A transfected m1 muscarinic acetylcholine receptor stimulates adenylate cyclase via phosphatidylinositol hydrolysis.

The m1 muscarinic acetylcholine receptor gene was transfected into and stably expressed in A9 L cells. The muscarinic receptor agonist, carbachol, stimulated inositol phosphate generation, arachidonic acid release, and cAMP accumulation in these cells. Carbachol stimulated arachidonic acid and inositol phosphate release with similar potencies, while cAMP generation required a higher concentration. Studies were performed to determine if the carbachol-stimulated cAMP accumulation was due to direct coupling of the m1 muscarinic receptor to adenylate cyclase via a GTP binding protein or mediated by other second messengers. Carbachol failed to stimulate adenylate cyclase activity in A9 L cell membranes, whereas prostaglandin E2 did, suggesting indirect stimulation. The phorbol ester, phorbol 12-myristate 13-acetate (PMA), stimulated arachidonic acid release yet inhibited cAMP accumulation in response to carbachol. PMA also inhibited inositol phosphate release in response to carbachol, suggesting that activation of phospholipase C might be involved in cAMP accumulation. PMA did not inhibit prostaglandin E2-, cholera toxin-, or forskolin-stimulated cAMP accumulation. The phospholipase A2 inhibitor eicosatetraenoic acid and the cyclooxygenase inhibitors indomethacin and naproxen had no effect on carbachol-stimulated cAMP accumulation. Carbachol-stimulated cAMP accumulation was inhibited with TMB-8, an inhibitor of intracellular calcium release, and W7, a calmodulin antagonist. These observations suggest that carbachol-stimulated cAMP accumulation does not occur through direct m1 muscarinic receptor coupling or through the release of arachidonic acid and its metabolites, but is mediated through the activation of phospholipase C. The generation of cytosolic calcium via inositol 1,4,5-trisphosphate and subsequent activation of calmodulin by m1 muscarinic receptor stimulation of phospholipase C appears to generate the accumulation of cAMP.