Chen H , Vandorpe DH , Xie X , Alper SL , Zeidel ML , Yu W .

R21 AG064633 NIA NIH HHS , U18 DA052346 NIDA NIH HHS

             calcium ion export across plasma membrane
             L-type voltage-gated calcium channel complex

                substance abuse
                bladder disease

	Fig. 1. Ketamine inhibits BSM contraction.a Representative traces of mouse BSM contraction in response to EFS (1, 2, 5, 10, 20, 50 Hz), showing dose-dependent inhibition by ketamine (0.1–500 µg ml−1). b Summarized data from experiments as in a (n = 12 BSM strips). Data are presented as mean values ± SD. c–e Representative traces of mouse BSM treated first with ketamine plus either carbachol (c n = 14 BSM strips), α,β-meATP (d n = 10 BSM strips), or 50 mM KCl (e n = 11 BSM strips), followed by drug washout and subsequent re-exposure to the indicated drugs in the absence of ketamine. f–h Summarized data from c–e. Data are shown as box and whiskers, center line is the median of the data set, box represents 75% of the data, and bars indicates whiskers from minimum to maximum. Data were analysed by one-way ANOVA with Bonferroni’s posthoc tests. P < 0.05 is considered to be significant and P values are given above the bars. Source data are provided as a Source Data file.
	Fig. 2. Ketamine inhibition of BSM contraction is not mediated by NMDAR.Representative filters show UV light-illuminated urine spots from a wild type mouse (a n = 21 filters) and a smooth muscle-specific NR1 knockout (SMNR1KO) mouse (b n = 18 filters). Summarized quantitative data (c) indicate normal voiding volume, normal spot number per void, and normal spot size for SMNR1KO mice. ± in c is SD of the mean. In b 400 mm2 white box at bottom right serves as area standard. d–g BSM contraction in SMNR1KO mice stimulated by EFS (d wild type n = 8 BSM strips, SMNR1KO n = 8 BSM strips), by 10 µM carbachol (e wild type n = 8 BSM strips, SMNR1KO n = 8 BSM strips), by 10 µM α,β-meATP (f wild type n = 8 BSM strips, SMNR1KO n = 8 BSM strips), or by 50 mM KCl (g wild type n = 8 BSM strips, SMNR1KO n = 8 BSM strips). Data are shown as box and whiskers, center line is the median of the data set, box represents 75% of the data, and bars indicates whiskers from minimum to maximum. Data were analysed by Student’s t-test. h ketamine concentration-dependent inhibition of contraction of BSM from SMNR1KO mice (n = 12 BSM strips). The data indicate that NMDAR does not mediate ketamine-induced inhibition of BSM contraction. Data are presented as mean values ± SD. Source data are provided as a Source Data file.
	Fig. 3. Ketamine and Cav1.2 agonist Bay k8644 are mutual antagonists.a Representative traces of Bay k8644-mediated potentiation of EFS-stimulated BSM contraction, summarized in d (n = 7 BSM strips). b Representative traces of Bay k8644-mediated potentiation of EFS-stimulated BSM contraction fully inhibited by ketamine and by nifedipine in dose-dependent manners, summarized in e (n = 8 BSM strips) and f (n = 12 BSM strips). c representative traces of ketamine-induced and nifedipine-induced inhibition of EFS-stimulated BSM contraction rescued by 50 or 200 nM Bay k8644, summarized in g (n = 17 BSM strips) and h (n = 13 BSM strips). Data are presented as mean values ± SD. These data support ketamine as an inhibitor of Cav1.2-mediated BSM contraction. Source data are provided as a Source Data file.
	Fig. 4. Ketamine inhibits Cav1.2-mediated calcium channel activity.a–c are representative Ba2+ current traces of two microelectrode voltage-clamped Xenopus oocytes, previously injected with water (a, n = 5 oocytes) or with cRNA encoding Cav1.2 subunits, recorded in the absence (b, n = 10 oocytes), or presence of ketamine (c n = 10 oocytes). d, e summarize data from a–c showing dose-dependent inhibition of Cav1.2-mediated Ba2+ current by ketamine (d n = 10 oocytes) or nifedipine (e n = 5 oocytes). f, g are representative whole cell current traces of fresh isolated BSM cells in the absence (f n = 8 BSM cells) or presence (g n = 8 BSM cells) of 100 µg ml−1 ketamine. h Summarizes data showing ketamine inhibition on Cav1.2-mediated Ba2+ current in freshly isolated mouse BSM cells as shown in f, g. Data are presented as mean values ± SD. Source data are provided as a Source Data file.
	Fig. 5. Ketamine inhibits Cav1.2-mediated calcium influx in mouse BSM cells.a–c Representative Fluo-4 Ca2+ images of primary cultured mouse BSM cells treated without (a) or with Bay k8644 (10 nM) (b), or treated first with ketamine (100 µg ml−1) and then with added Bay k8644 (10 nM) (c). Scale bar: 40 µm. d Nonlinear curve fit of Bay k8644 concentration-dependent increase in mouse BSM cell [Ca2+]i with EC50 of 9.6 nM (n = 10, 23, 18, 19, 10, and 29 BSM cells for respective [Bay k8644] = 0, 0.1, 1, 10, 200, and 1000 nM). Bars indicate the SD of the means. e 10 nM Bay k8644-induced increase in BSM intracellular [Ca2+] (n = 39 BSM cells) was inhibited by 100 µg ml−1 ketamine (n = 25 BSM cells)) and by 10 µM nifedipine (n = 39 BSM cells). f Hundred millimolar of KCl-stimulated increase in BSM intracellular [Ca2+] (n = 39) was inhibited by 100 µg ml−1 ketamine (n = 25 BSM cells) and by 10 µM nifedipine (n = 39 BSM cells). g Hundred micromolar of carbachol-stimulated increase in BSM intracellular [Ca2+] (n = 27 BSM cells) was inhibited by 100 µg ml−1 ketamine (n = 27 BSM cells), and by 10 µM nifedipine (n = 30 BSM cells). h Hundred micromolar of ATP-stimulated increase in BSM intracellular [Ca2+] (n = 43 BSM cells) was inhibited by 100 µg ml−1 ketamine (n = 22 BSM cells) and by 10 µM nifedipine (n = 21 BSM cells). Data are shown as box and whiskers, center line is the median of the data set, box represents 75% of the data, and bars indicates whiskers from minimum to maximum. Data were analysed by two tailed Student’s t-test. P < 0.05 is considered to be significant and P values are given above the bars. Source data are provided as a Source Data file.
	Fig. 6. Ketamine reduces Cav1.2-stimulated mRNA and protein levels in BSM cells.Mouse BSM strips were subjected to 50 mM KCl activation of Cav1.2 for 0–120 min and then lysed for mRNA preparation. Changes in c-fos (a n = 9 BSM strips) and c-jun (b n = 9 BSM strips) mRNA were measured by quantitative RT-PCR. Upregulation of both c-fos and c-jun were inhibited by 100 µg ml−1 ketamine and by 10 µM nifedipine. c Representative immunoblot from two independent experiments showing that upregulation of c-fos and c-jun by 30 min treatment with 50 mM KCl to activate Cav1.2 was inhibited by ketamine (100 µg ml−1) and by nifedipine (10 µg ml−1). d summarized data (n = 5 BSM strips) of experiments similar to that shown in c. e 50 mM KCl upregulated expression and nuclear translocation of c-fos and c-jun in human BSM cells in a manner inhibited by ketamine (100 µg ml−1) and rescued by Bay k8644 (200 nM). Scale bar: 20 µm. These images, representative of two separate triplicate experiments, are summarized in f (n = 38–46 BSM cells). Data are shown as box and whiskers, center line is the median of the data set, box represents 75% of the data, and bars indicates whiskers from minimum to maximum. Data are analysed by two tailed Student’s t-test. P < 0.05 is considered to be significant and P values are given above the bars. Source data are provided as a Source Data file.
	Fig. 7. Ketamine and nifedipine dose-dependently inhibit human BSM cell proliferation.Primary cultured human BSM cells were incubated with ketamine or nifedipine for 1–7 days at the indicated concentrations, cell proliferation was assessed by alamar blue assay in the presence of ketamine (a n = 4) or nifedipine (b n = 4). Ketamine and nifedipine inhibited hBSM cell proliferation. c Immunofluorescent staining of ki67 (green) in hBSM cells was inhibited by pretreatment with ketamine (100 µg ml−1) or nifedipine (10 µM), but inhibition was rescued by 200 nM Bay k8644, representative images are from two independent experiments with at least three samples each group at each experiment. Scale bar: 40 µm. Data are presented as mean values ± SD. Source data are provided as a Source Data file.
	Fig. 8. SMCav1.2+/− mice exhibit abnormal BSM morphology and contractility.a, b Representative images from three independent experiments: Masson’s trichrome stained bladder sections show that SMCav1.2+/- mouse bladders are smaller with a thinner BSM layer. Scale bars are, from left to right, 400 µm, 200 µm, and 20 µm, respectively. The summarized data are shown in c–f. c, d (n = 8 mice for both wildtype and SMCav1.2+/− groups) show that SMCav1.2+/− mice have lower body weight and bladder weight. e, f (n = 6 mice for both wildtype and SMCav1.2+/− groups) show that SMCav1.2+/− mice have reduced bladder wall and correspondingly lower muscle layer thickness. Individual BSM cells were subjected to immunofluorescence staining and imaged with antiβ1 integrin antibody (green) and DAPI for nuclei (blue). BSM cell cross sectional area was quantitated using Fiji software. SMCav1.2+/− mice BSM cell size was smaller (g n = 32, 54, 55, and 73 cells for wildtype female, SMCav1.2+/− female, wildtype male, and SMCav1.2+/− male mice groups, respectively). h, i Representative BSM contraction force traces in response to EFS show that reduced contraction force measured in SMCav1.2+/− BSM tissue, was enhanced by Bay k8644. j–m summarized data showing SMCav1.2+/− mice BSM strips exhibited decreased contraction force in response to EFS stimulation (j n = 9 BSM strips for both wildtype and SMCav1.2+/− groups), in response to 50 mM KCl (k n = 9 BSM strips for both groups), in response to 10 µM carbachol (l n = 11 BSM strips for both groups), and in response to 10 µM α,β-meATP (m n = 10 BSM strips for both groups). In each condition, contraction was significantly enhanced by 200 nM Bay k8644. Data are shown as box and whiskers, center line is the median of the data set, box represents 75% of the data, and bars indicates whiskers from minimum to maximum. Data are analysed by two tailed Student’s t-test. P < 0.05 is considered to be significant and P values are given above the bars. Source data are provided as a Source Data file.
	Fig. 9. SMCav1.2+/− mice exhibit altered urodynamics mimicking ketamine cystitis.a–c Representative VSA images from SMCav1.2+/− mice showing increased number of voids and smaller void size, both corrected by Bay k8644 (2 mg/kg i.p.). e–g (n = 31 filters for wild type, and n = 18 filters for SMCav1.2+/− with or without Bay k8644), summarized data indicating SMCav1.2+/− mice have increased numbers of primary voiding spots (PVS: voiding spot area ≥80 mm2) and reduced PVS size (f). d CMG traces from SMCav1.2+/− mice. h–i (n = 5 mice) summarized voiding interval (h), basal pressure (i), threshold pressure (j), peak pressure (k), and compliance (l), which were quantitated by paired t-test. SMCav1.2+/− mice showed reduced voiding interval, smaller voiding size, reduced peak pressure and compliance, each rescued by infusion of Bay k8644 (200 nM). Data are shown as box and whiskers, center line is the median of the data set, box represents 75% of the data, and bars indicates whiskers from minimum to maximum. Data are analysed by two tailed Student’s t-test (e–g) and two tailed paired t-test (h–l). P < 0.05 is considered to be significant and P values are given above the bars. Source data are provided as a Source Data file.
	Fig. 10. Ketamine and nifedipine regulated mouse urodynamics resembling ketamine cystitis in humans.a, b Representative CMG traces of ketamine cystitis-like voiding phenotypes induced by intravesical ketamine (a 100, 500 µg ml−1) and nifedipine (b 10, 100 µM) are reversed by Bay k8644 infusion (200 nM). Summarized CMG data (c–l n = 5 mice for ketamine group and n = 7 mice for nifedipine group) show that decreased voiding interval (c, h), peak pressure (f, k), and compliance (g, l), were each rescued by Bay k8644 infusion. Data are shown as box and whiskers, center line is the median of the data set, box represents 75% of the data, and bars indicates whiskers from minimum to maximum. Data were analysed by paired two tail Student’s t-tests. P < 0.05 is considered to be significant and P values are given above the bars. Source data are provided as a Source Data file.

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