Efficacy of Oral SERDs in the treatment of ER+, HER2 - metastatic breast cancer, a stratified analysis of the ESR1 wild type and mutant subgroups.

BACKGROUND: Oral SERDs are a novel drug class that have been developed to counteract resistance due to ESR1 mutations. Several SERDs have emerged from phase 2 and 3 trials, with the FDA limiting approval for Elacestrant to patients with ESR1mt tumours despite PFS benefit in the overall population. However, questions remain on whether patients with ESR1wt tumours stand to benefit from oral SERDs. PATIENTS AND METHODS: Manuscripts and conference presentations of Randomised Controlled Trials were extracted after a systematic search of Embase, PubMed and Cochrane from inception until January 21,2023. RCTs investigating the efficacy of oral SERDs versus endocrine therapy for ER positive, HER2 negative advanced breast cancer, and which reported the Kaplan Meier (KM) curves of PFS in the overall and ESR1 mutant (ESR1mt) population were selected. A graphical reconstructive algorithm was applied to estimate time-to-event outcomes from reported KM curves in all overall and ESR1mt cohorts. A bipartite matching algorithm, KMSubtraction, was used to derive survival data for unreported (ESR1wt) subgroups. An individual patient data (IPD) meta-analysis was then pursued, pooling data by ESR1 mutation status in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) and Cochrane Guidelines for IPD. RESULTS: The randomized clinical trials ACELERA, AMEERA-3, EMERALD and SERENA-2 were included, totalling 1290 patients. In the pooled analysis of the overall cohort, PFS benefit was observed with oral SERDs when compared with treatment of physicians choice (TPC) (HR 0.783, 95%CI 0.681-0.900, p<0.001). In the ESR1mt subgroup, oral SERDs demonstrated improved PFS (HR 0.557, 95%CI 0.440-0.705, p<0.001) compared to TPC. In the ESR1wt subgroup, oral SERDs demonstrated no significant PFS benefit (HR 0.944, 95%CI 0.783-1.138, p=0.543) when compared to TPC. CONCLUSIONS: The results of this IPD meta-analysis suggests that PFS benefit in the overall population is mainly driven by the ESR1mt subgroup.

Cosmic-ray antinuclei as messengers of new physics: status and outlook for the new decade.

The precise measurement of cosmic-ray antinuclei serves as an important means for identifying the nature of dark matter and other new astrophysical phenomena, and could be used with other cosmic-ray species to understand cosmic-ray production and propagation in the Galaxy. For instance, low-energy antideuterons would provide a "smoking gun" signature of dark matter annihilation or decay, essentially free of astrophysical background. Studies in recent years have emphasized that models for cosmic-ray antideuterons must be considered together with the abundant cosmic antiprotons and any potential observation of antihelium. Therefore, a second dedicated Antideuteron Workshop was organized at UCLA in March 2019, bringing together a community of theorists and experimentalists to review the status of current observations of cosmic-ray antinuclei, the theoretical work towards understanding these signatures, and the potential of upcoming measurements to illuminate ongoing controversies. This review aims to synthesize this recent work and present implications for the upcoming decade of antinuclei observations and searches. This includes discussion of a possible dark matter signature in the AMS-02 antiproton spectrum, the most recent limits from BESS Polar-II on the cosmic antideuteron flux, and reports of candidate antihelium events by AMS-02; recent collider and cosmic-ray measurements relevant for antinuclei production models; the state of cosmic-ray transport models in light of AMS-02 and Voyager data; and the prospects for upcoming experiments, such as GAPS. This provides a roadmap for progress on cosmic antinuclei signatures of dark matter in the coming years.

TPM3 deletions cause a hypercontractile congenital muscle stiffness phenotype.

OBJECTIVE: Mutations in TPM3, encoding Tpm3.12, cause a clinically and histopathologically diverse group of myopathies characterized by muscle weakness. We report two patients with novel de novo Tpm3.12 single glutamic acid deletions at positions ΔE218 and ΔE224, resulting in a significant hypercontractile phenotype with congenital muscle stiffness, rather than weakness, and respiratory failure in one patient. METHODS: The effect of the Tpm3.12 deletions on the contractile properties in dissected patient myofibers was measured. We used quantitative in vitro motility assay to measure Ca(2+) sensitivity of thin filaments reconstituted with recombinant Tpm3.12 ΔE218 and ΔE224. RESULTS: Contractility studies on permeabilized myofibers demonstrated reduced maximal active tension from both patients with increased Ca(2+) sensitivity and altered cross-bridge cycling kinetics in ΔE224 fibers. In vitro motility studies showed a two-fold increase in Ca(2+) sensitivity of the fraction of filaments motile and the filament sliding velocity concentrations for both mutations. INTERPRETATION: These data indicate that Tpm3.12 deletions ΔE218 and ΔE224 result in increased Ca(2+) sensitivity of the troponin-tropomyosin complex, resulting in abnormally active interaction of the actin and myosin complex. Both mutations are located in the charged motifs of the actin-binding residues of tropomyosin 3, thus disrupting the electrostatic interactions that facilitate accurate tropomyosin binding with actin necessary to prevent the on-state. The mutations destabilize the off-state and result in excessively sensitized excitation-contraction coupling of the contractile apparatus. This work expands the phenotypic spectrum of TPM3-related disease and provides insights into the pathophysiological mechanisms of the actin-tropomyosin complex.

Pharmacogenomic diversity in Singaporean populations and Europeans.

Differences in the frequency of pharmacogenomic variants may influence inter-population variability in drug efficacy and risk of adverse drug reactions (ADRs). We investigated the diversity of ∼ 4500 genetic variants in key drug-biotransformation and -response genes among three South East Asian populations compared with individuals of European ancestry. We compared rates of reported ADRs in these Asian populations to determine if the allelic differentiation corresponded to an excess of the associated ADR. We identified an excess of ADRs related to clopidogrel in Singaporean Chinese, consistent with a higher frequency of a known risk variant in CYP2C19 in that population. We also observed an excess of ADRs related to platinum compounds in Singaporean CHS, despite a very low frequency of known ADR risk variants, suggesting the presence of additional genetic and non-genetic risk factors. Our results point to substantial diversity at specific pharmacogenomic loci that may contribute to inter-population variability in drug response phenotypes.

Exploring factors influencing outpatient radiology attendance based on Health Belief Model (HBM): A qualitative study.

INTRODUCTION: Non-attendance for radiology outpatient appointments is a global issue and is associated with adverse clinical outcomes and operational inefficiencies. This paper aims to understand the underlying factors influencing outpatient radiology attendances based on the Health Belief Model (HBM). METHODS: Purposive sampling was used to recruit patients (n=30) for in-depth semi-structured telephone interviews. Inclusion criteria comprised participants who were above 21 years old and fluent in English, while participants reliant on third-party assistance (e.g., nursing homes and prison services), to attend the appointment were excluded. The interviews were recorded and transcribed verbatim. The COREQ (Consolidated Criteria for Reporting Qualitative Research) was utilised to guide the reporting of this study. The data analysis involved a hybrid thematic analysis approach using NVivo. RESULTS: Six key themes associated with appointment adherence in radiology were identified. These themes included: 1) prioritising health and acceptance of current medical conditions; 2) the impact of perceived severity on non-attendance; 3) perceived benefits of attending appointments; 4) perceived barriers to attendance; 5) influential prompts; and 6) confidence in attendance. CONCLUSION: This is the first study of its kind to utilise the HBM to examine factors influencing attendance adherence among radiology outpatients in Singapore. Costs, prompts, and the perceived severity of the condition play pivotal roles in shaping the health-seeking behaviours of these outpatients while professionalism of healthcare staff and barriers to attendance present opportunities for providers to address patients' lack of interest towards their appointments. IMPLICATIONS FOR PRACTICE: Findings of this study will contribute to the development of personalised, targeted interventions for improving patient engagement and attendance in radiology settings.

Replication of 13 obesity loci among Singaporean Chinese, Malay and Asian-Indian populations.

OBJECTIVE: Recent genome-wide association studies (GWAS) have identified 38 obesity-associated loci among European populations. However, their contribution to obesity in other ethnicities is largely unknown. METHODS: We utilised five GWAS (N=10 482) from Chinese (three cohorts, including one with type 2 diabetes and another one of children), Malay and Indian ethnic groups from Singapore. Data sets were analysed individually and subsequently in combined meta-analysis for Z-score body-mass index (BMI) associations. RESULTS: Variants at the FTO locus showed the strongest associations with BMI Z-score after meta-analysis (P-values 1.16 × 10(-7)-7.95 × 10(-7)). We further detected associations with nine other index obesity variants close to the MC4R, GNPDA2, TMEM18, QPCTL/GIPR, BDNF, ETV5, MAP2K5/SKOR1, SEC16B and TNKS/MSRA loci (meta-analysis P-values ranging from 3.58 × 10(-4)-1.44 × 10(-2)). Three other single-nucleotide polymorphisms (SNPs) from CADM2, PTBP2 and FAIM2 were associated with BMI (P-value ≤ 0.0418) in at least one dataset. The neurotrophin/TRK pathway (P-value=0.029) was highlighted by pathway-based analysis of loci that had statistically significant associations among Singaporean populations. CONCLUSION: Our data confirm the role of FTO in obesity predisposition among Chinese, Malays and Indians, the three major Asian ethnic groups. We additionally detected associations for 12 obesity-associated SNPs among Singaporeans. Thus, it is likely that Europeans and Asians share some of the genetic predisposition to obesity. Furthermore, the neurotrophin/TRK signalling may have a central role for common obesity among Asians.

Utility and safety of procalcitonin in an antimicrobial stewardship program (ASP) in patients with malignancies.

As data on procalcitonin utility in antibiotics discontinuation [under an antimicrobial stewardship program (ASP)] in patients with malignancies are lacking, we aimed to evaluate the utility of procalcitonin in an ASP in patients with malignancies. We conducted a retrospective review of the ASP database of all patients with malignancies in whom at least one procalcitonin level was taken and our ASP had recommended changes in carbapenem regimen, from January to December 2011. We compared clinical outcomes between two groups of patients: patients whose physicians accepted and those whose physicians rejected ASP interventions. There were 749 carbapenem cases reviewed. Ninety-nine were suggested to either de-escalate, discontinue antibiotics, or narrow the spectrum of empiric treatment, based on procalcitonin trends. While there was no statistical difference in the mortality within 30 days post-ASP intervention (accepted: 8/65 patients vs. rejected: 9/34 patients; p = 0.076), the median duration of carbapenem therapy was significantly shorter (5 vs. 7 days; p = 0.002). Procalcitonin use safely facilitates decisions on antibiotics discontinuation and de-escalation in patients with malignancies in the ASP.

Extradural haematoma secondary to brown snake (Pseudonaja species) envenomation.

A 4-year-old Siberian Husky dog was treated with brown snake antivenom by his regular veterinarian after a witnessed episode of brown snake envenomation. The dog was discharged 5 hours post presentation despite an ongoing coagulopathy. The dog was presented to the emergency centre 2 hours later because the owner believed the dog to be in pain. Initial examination revealed an ambulatory but neurologically normal patient with thoracolumbar pain and laboratory evidence of a coagulopathy. Despite correction of the coagulopathy, the signs progressed to bilateral hind limb paresis after approximately 3 hours of hospitalisation, and continued to deteriorate over the next 56 hours to loss of deep pain perception in the right hind limb. Computed tomography imaging identified the presence of an extradural haematoma which was subsequently removed via a hemilaminectomy. Surgical decompression was successful in treating the spinal compression and the dog recovered with minimal complications. To our knowledge this is the first report of extradural haematoma secondary to coagulopathy induced by brown snake envenomation.

Evaluation of 3D modality-independent elastography for breast imaging: a simulation study.

This paper reports on the development and preliminary testing of a three-dimensional implementation of an inverse problem technique for extracting soft-tissue elasticity information via non-rigid model-based image registration. The modality-independent elastography (MIE) algorithm adjusts the elastic properties of a biomechanical model to achieve maximal similarity between images acquired under different states of static loading. A series of simulation experiments with clinical image sets of human breasts were performed to test the ability of the method to identify and characterize a radiographically occult stiff lesion. Because boundary conditions are a critical input to the algorithm, a comparison of three methods for semi-automated surface point correspondence was conducted in the context of systematic and randomized noise processes. The results illustrate that 3D MIE was able to successfully reconstruct elasticity images using data obtained from both magnetic resonance and x-ray computed tomography systems. The lesion was localized correctly in all cases and its relative elasticity found to be reasonably close to the true values (3.5% with the use of spatial priors and 11.6% without). In addition, the inaccuracies of surface registration performed with thin-plate spline interpolation did not exceed empiric thresholds of unacceptable boundary condition error.

Validation of hyperbaric oxygen treatment software for use with monoplace chambers.

Hyperbaric oxygen (HBO2) therapy is increasingly used in the treatment of a wide variety of medical conditions. However, for monoplace chambers, there is some uncertainty when sufficiently high oxygen concentrations are attained, because most chambers are not instrumented to measure oxygen. To remedy this, Microsoft Excel-based software, HBO O2 Smart Guide, was developed to simulate the atmosphere ofmonoplace chambers during treatment. Based upon chamber dimensions, patient weight, oxygen purge rates, desired pressurization, and HBO2 time, the program calculates oxygen concentration, consumption and exposure for each treatment. Software testing was conducted using four different chambers instrumented with an oxygen analyzer. Two purge rate profiles were used: constant, and biphasic (a high initial purge rate was changed to a lower plateau rate when pressurization was reached). Comparison of measured and calculated times to reach 95% oxygen concentration within the chambers demonstrated the software was accurate within 1%. The HBO O2 Smart Guide enables optimum purge profiles to be simulated with resultant potential improvements in HBO2 treatment efficacy, calculation of effective oxygen exposures (actual time during prescribed treatment during which patient breathes > or = 95% oxygen) to enable more accurate comparison of treatment profiles and outcomes, and cost savings in oxygen usage. This software will enable clinicians to provide more consistent HBO2 treatments.

Variants in ACTG2 underlie a substantial number of Australasian patients with primary chronic intestinal pseudo-obstruction.

BACKGROUND: Primary chronic intestinal pseudo-obstruction (CIPO) is a rare, potentially life-threatening disorder characterized by severely impaired gastrointestinal motility. The objective of this study was to examine the contribution of ACTG2, LMOD1, MYH11, and MYLK mutations in an Australasian cohort of patients with a diagnosis of primary CIPO associated with visceral myopathy. METHODS: Pediatric and adult patients with primary CIPO and suspected visceral myopathy were recruited from across Australia and New Zealand. Sanger sequencing of the genes encoding enteric gamma-actin (ACTG2) and smooth muscle leiomodin (LMOD1) was performed on DNA from patients, and their relatives, where available. MYH11 and MYLK were screened by next-generation sequencing. KEY RESULTS: We identified heterozygous missense variants in ACTG2 in 7 of 17 families (~41%) diagnosed with CIPO and its associated conditions. We also identified a previously unpublished missense mutation (c.443C>T, p.Arg148Leu) in one family. One case presented with megacystis-microcolon-intestinal hypoperistalsis syndrome in utero with subsequent termination of pregnancy at 28 weeks' gestation. All of the substitutions identified occurred at arginine residues. No likely pathogenic variants in LMOD1, MYH11, or MYLK were identified within our cohort. CONCLUSIONS AND INFERENCES: ACTG2 mutations represent a significant underlying cause of primary CIPO with visceral myopathy and associated phenotypes in Australasian patients. Thus, ACTG2 sequencing should be considered in cases presenting with hypoperistalsis phenotypes with suspected visceral myopathy. It is likely that variants in other genes encoding enteric smooth muscle contractile proteins will contribute further to the genetic heterogeneity of hypoperistalsis phenotypes.

The contribution of recently identified adult BMI risk loci to paediatric obesity in a Singaporean Chinese childhood dataset.

INTRODUCTION: Recent genome-wide association studies have identified 103 adult obesity risk loci; however, it is unclear if these findings are relevant to East-Asian childhood body mass index (BMI) levels. METHODS AND RESULTS: We evaluated for paediatric obesity associations at these risk loci utilizing genome-wide data from Chinese childhood subjects in the Singapore Cohort study Of the Risk factors for Myopia study (N = 1006). A weighted gene-risk score of all adult obesity risk loci in the Singapore Cohort study Of the Risk factors for Myopia study showed strong associations with BMI at age 9 (p-value = 3.40 × 10-12 ) and 4-year average BMI (age 9 to 12, p-value = 6.67 × 10-8 ). Directionally consistent nominal associations for 15 index single nucleotide polymorphisms (SNPs) (p-value < 0.05) were observed. Pathway analysis with genes from these 15 replicating loci revealed over-representation for the G-protein-coupled receptor (GPCR)-mediated integration of entero-endocrine signalling pathway exemplified by L-cell (adjusted p-value = 0.018). Evaluations of birth weight to modify the effects of BMI risk SNPs in paediatric obesity did not reveal significant interactions, and these SNPs were generally not associated with birth weight. CONCLUSIONS: At least some common adult BMI risk variants predispose to paediatric obesity risk in East-Asians.

Expression of an amphibian homolog of the Eph family of receptor tyrosine kinases is developmentally regulated.

In order to study the function of tyrosine kinase receptors during Xenopus development, we have isolated Xek (Xenopus Elk-like kinase), a tyrosine kinase receptor, which shows significant homology to rat Elk and chicken cek5, members of the Eph family. Xek exists as a maternally expressed mRNA which decreases in expression at the mid blastula transition and reappears at late neurulation in Xenopus. Xek mRNA is expressed at higher levels in the anterior and dorsal regions of embryonic stages 16, 24 and 37. In adult Xenopus tissues, Xek appears to be ubiquitously expressed with higher expression observed in brain and ovary. In situ hybridization analysis demonstrates localized mRNA expression in the brain, brachial arches, trigeminal facial ganglion, and the retina of the swimming tadpole stage of development. The similarities in sequence and expression pattern suggest that Xek is an amphibian member of the Eph family and may play a role in the development or function of the central nervous system.

High-resolution 13C nuclear magnetic resonance studies of small unilamellar vesicles containing glycophorin A.

Glycophorin A, the major sialoglycoprotein of the human erythrocyte membrane, has been incorporated in small unilamellar vesicles containing phosphatidylcholine and phosphatidylethanolamine in varying proportions. Hydrocarbon chains of these two lipids have been selectively enriched with 13C and 13C-NMR spin relaxation parameters have been monitored in the presence and absence of protein. Perturbations to 13C line-widths and spin-lattice relaxation times are found to be small and consistent with relatively weak interactions. The perturbations, though small, show some specificity. The carbonyl carbons in both phosphatidylcholine and phosphatidylethanolamine are broadened, but in addition the olefinic carbons in phosphatidylethanolamine are broadened.

Human tumor fluorouracil trapping: clinical correlations of in vivo 19F nuclear magnetic resonance spectroscopy pharmacokinetics.

We previously reported that fluorouracil (5FU) accumulation and metabolism in human livers and tumors can be studied by in vivo nuclear magnetic resonance spectroscopy (NMRS). We have extended these observations by evaluating the pharmacokinetics of 5FU in the tumors of 11 patients with carcinoma of the breast, colon, endometrium, cervix, and kidney, using 19F-NMRS in a 1.5 Magnetom (Siemens Medical Systems, Cerrito, CA) magnetic resonance imaging unit (MRI). These NMRS measurements detected a long-lived tumor pool of 5FU in six of 11 tumors in our patients including carcinomas in the pelvis, breast, lung, and liver. The half-life (T1/2) of this tumor pool of "trapped" 5FU was 0.33 to 1.3 hours (20 to 78 minutes), much longer than the T1/2 of 5FU in blood (5 to 15 minutes). Neither the anabolites of 5FU (fluorinated nucleosides, nucleotides, 5FU-RNA, or 5FU-thymidylate synthase) nor the catabolites (eg, fluorobetaalanine [FBAL]) were detectable by 19F NMRS. Patient response to chemotherapy appeared to correlate with the extent of trapping of free 5FU in the human tumors: in the seven patients receiving 5FU, or 5FU or FUdR plus leucovorin, four of four patients whose tumors trapped 5FU responded to fluorinated pyrimidine chemotherapy, whereas three patients in whom there was a failure to detect tumor trapping were resistant to 5FU. We conclude that NMRS is clinically feasible, and enables investigators to study 5FU pharmacokinetics and metabolism in tumors in vivo. 19F-NMRS of 5FU allows for in vivo evaluation of 5FU metabolic modulation and might be able to guide therapeutic decisions.

Adenine nucleotide metabolism in hearts of diabetic rats. Comparison to diaphragm, liver, and kidney.

High-performance liquid chromatography analysis of acid-extracted tissues revealed decreases of high-energy nucleotides and increases in low-energy nucleotides and metabolites in heart, diaphragm, and liver but not in kidneys of diabetic rats. In comparison with nondiabetic rats, the total adenine nucleotide content of diabetic rat heart and diaphragm but not liver decreased, indicating an increase in catabolism of AMP. Maximal initial rates of the AMP catabolic enzymes 5'-nucleotidase, adenosine deaminase, and AMP deaminase were elevated in the hearts of BB/Wistar and streptozocin-induced diabetic rats. Nucleotide salvage enzymes adenylosuccinate synthetase and adenylosuccinate lyase were elevated above normal in the diabetic heart, whereas hypoxanthine-guanine phosphoribosyl transferase was not altered. Cytosolic-to-mitochondrial ratios from maximal initial rates after correction for mitochondrial breakage were increased above controls in diabetic hearts for nucleoside diphosphokinase and aspartate aminotransferase. Nucleotide levels, degradation rates, and substrate compartmentation between cytosol and mitochondria are discussed in relation to concurrent diabetes.

Inhibitory effect of phorbol ester on bradykinin-induced phosphoinositide hydrolysis and calcium mobilization in cultured canine tracheal smooth muscle cells.

Regulation of the increase in inositol 1,4,5-trisphosphate (IP3) production and intracellular Ca2+ concentration ([Ca2+]i) by protein kinase C (PKC) was investigated in cultured canine tracheal smooth muscle cells (TSMCs). Stimulation of TSMCs by bradykinin (BK) led to IP3 formation and caused an initial transient peak followed by a sustained elevation of [Ca2+]i in a concentration-dependent manner. Pretreatment of TSMCs with phorbol 12-myristate 13-acetate (PMA, 1 microM) for 30 min blocked the BK-induced IP3 formation and Ca2+ mobilization. However, this inhibition was reduced after incubating the cells for 4 h with PMA. Inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate at 1 microM, did not inhibit these responses to BK. Prior treatment with staurosporine (1 microM), a PKC inhibitor, inhibited the effect of PMA on the BK-induced response, suggesting that the effect of PMA is mediated by the activation of PKC. In parallel experiments, a change of PKC activity was observed. PMA rapidly decreased PKC activity in the cytosol of TSMCs, while increasing it transiently in the cell membranes within 30 min. Thereafter the membrane-associated PKC activity decreased and persisted for at least 24 h of PMA treatment. Moreover, treatment with 1 microM PMA for 2 and 24 h did not significantly change the KD and Bmax of the BK receptor for [H]BK binding (control: KD = 2.3 +/- 0.3 nM, Bmax = 25.2 +/- 1.4 fmol/mg protein). These results suggest that activation of PKC inhibit IP3 accumulation and consequently attenuate [Ca2+]i increase or inhibit independently both responses. The PMA-induced inhibition of responses to BK was associated with an increase in membranous PKC activity.

5-Hydroxytryptamine-stimulated calcium mobilization in cultured canine tracheal smooth muscle cells.

5-Hydroxytryptamine (5-HT)-induced increase of intracellular Ca2+ concentration ([Ca2+]i) was monitored in cultured canine tracheal smooth muscle cells (TSMCs) using a fluorescent Ca2+ indicator Fura-2. Stimulation of TSMCs by 5-HT produced an initial transient peak followed by a sustained, concentration-dependent elevation of [Ca2+]i. The log (EC50) values of 5-HT for the peak and sustained plateau responses were -7.43 and -7.60 M, respectively. 5-HT1A and 5-HT3 receptor antagonists, NAN-190 and metoclopramide, inhibited the 5-HT-stimulated increase in [Ca2+]i with pKB values of 6.3 and 6.2, respectively, indicating that the 5-HT receptors mediating Ca2+ signal had low affinity for these receptor antagonists. In contrast, 5-HT2A receptor antagonists, ketanserin and mianserin, had high affinity in antagonizing the changes in [Ca2+]i response to 5-HT with pKB values of 8.3 and 8.3, respectively. The sustained elevation of [Ca2+]i was dependent on the presence of extracellular Ca2+. Removal of extracellular Ca2+ by addition of 2 mM EGTA during the sustained phase caused a rapid decline in [Ca2+]i to the resting level. In the absence of extracellular Ca2+, only an initial peak was observed which then declined to the resting level; the sustained elevation of [Ca2+]i could then be evoked by addition of 1.8 mM Ca2+ in the continued presence of 5-HT. Ca2+ influx was required for the changes of [Ca2+]i, since the Ca(2+)-channel blockers, diltiazem, verapamil, and Ni2+, decreased both the initial and sustained elevation of [Ca2+]i in response to 5-HT. These Ca(2+)-channel blockers also decreased the sustained elevation of [Ca2+]i when applied during the plateau phase. In conclusion, these findings indicate that the initial increase in [Ca2+]i stimulated by 5-HT acting on 5-HT2A receptors is due to the release of Ca2+ from internal stores, followed by the influx of external Ca2+ into the cells. The influx of extracellular Ca2+ partially involves a diltiazem and verapamil sensitive Ca2+ channel.