Mast cells and degranulation of pre-formed inflammatory mediators contribute to lower urinary tract symptoms. This study investigates pathways by which the mast cell stimulator Compound 48/80 alters urinary bladder smooth muscle contractility via mast cell activation. We hypothesized that: (1) mast cell degranulation will cause spontaneous urinary bladder smooth muscle contractions; and (2) these contractions are caused by urothelium-derived PGE2. Urothelium intact and denuded urinary bladder strips were collected from mast cell sufficient (C57Bl/6) and deficient (B6.Cg-Kitw-sh) mice to determine if Compound 48/80 altered urinary bladder smooth muscle (UBSM) contractility. Electrical field stimulation was used to assess the effects of Compound 48/80 on nerve-evoked contractions. Antagonists/inhibitors were utilized to identify prostanoid signaling pathways activated or if direct activation of nerves was involved. Compound 48/80 caused slow-developing contractions, increased phasic activity, and augmented nerve-evoked responses in both mast cell sufficient and deficient mice. Nerve blockade had no effect on these responses; however, they were eliminated by removing the urothelium. Blocking P2 purinoreceptors, cyclooxygenases, or G protein signaling abolished Compound 48/80 responses. However, only combined blockade of prostaglandin E2 (EP1), prostaglandin F2α (FP), and thromboxane A2 (TP) receptors inhibited Compound 48/80 induced responses. Thus, the effects of compound 48/80 are urothelium dependent, but independent of mast cells. Further, these effects are mediated by druggable inflammatory pathways that may be used to manage inflammatory non-neurogenic bladder hyperactivity. Finally, these data strongly suggest that great care must be taken when using Compound 48/80 to determine mast cell-dependent responses in the urinary bladder.

ID 22897 Poster Board 30Inflammatory hyperalgesia induced by mast cell degranulation releases histamine within the urinary bladder that disrupts the micturition reflex and alters detrusor contractility. Psychological stress also causes lower urinary tract symptoms (frequency, urgency, and hesitancy) in 4-week-old mice with a pathophysiology similar to neurogenic inflammatory hyperalgesia. Most investigations have solely focused on molecular changes to nerve sensitivity instead of direct changes to urinary bladder smooth muscle activity. Recently, our lab found that the urinary bladder smooth muscle of unstressed mice contracted and rapidly desensitized to histamine in an H1 receptor-dependent manner. We hypothesized that social stress increases histamine H1 receptor mRNA expression to prolong histamine-induced urinary bladder smooth contractions.4-week-old C57BL/6 male (N=6) mice were randomly housed with CD1 adult male aggressor mice for 5 minutes, after which both mice were placed in barrier housing for 1 hour. Social stress was repeated with different aggressor mice for 14 days. Afterward, bladders from 6-week-old mice were dissected and cut into strips from non-stressed (N=3) and stressed (N=3) mice to measure isometric contractile force generation and changes in contractility by histamine (200 mM). Individual bladder smooth muscle cells were also dissociated and collected for single-cell qRT-PCR. Counter to our hypothesis, histamine-induced contractions were markedly reduced in stressed as compared to non-stressed mice. Surprisingly, however, single-cell qRT-PCR performed on isolated smooth muscle cells uncovered an upregulation of histamine H3 receptor mRNA in stressed mice that was absent in non-stressed mice. H3 receptors were present and constitutively active in the tissue, as the H3 receptor antagonist ciproxifan (10 mM) recovered the response to histamine in stressed bladders but had no effect in non-stressed bladders. In both conditions histamine-induced contractions rapidly desensitized, implying that H3 receptors do not affect this physiological mechanism. Our data aligns with previous findings that support a role for elevated histamine H3 receptor mRNA in patients with interstitial cystitis. Additionally, our data suggest that the histamine H3 receptor might have a protective role in social stress by preventing histamine-induced changes to smooth muscle contractility in stressed bladders.

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Mast cell degranulation and histamine release contribute to painful bladder syndrome, urinary tract infections, interstitial cystitis and other bladder disorders. The effects of histamine are well‐studied in other tissues, but little is known about the role of histamine and histamine receptors within the bladder. Since other smooth muscle cells express H1 (contractile) and H2 (relaxant) histamine receptors, we tested the hypothesis that histamine causes contraction of the urinary bladder smooth muscle in mice. All procedures followed institutional guidelines and were approved by the Institutional Animal Care and Use Committees of Michigan State University. Six to 12‐week‐old C57Bl/6 mice were euthanized and dissected bladders were cut into approximately 2 mm wide bladder strips, with or without the urothelium attached, for isometric contractility experiments. Both histamine H1 and H2 receptors were expressed in bladder smooth muscle cells. Increasing concentrations of histamine (100 nM – 300 μM) did not contract strips without urothelium and minimally contracted bladder strips with urothelium. However, bolus administration of 200 μM histamine caused a rapid but transient contraction in strips with and without urothelium. The H1 receptor blocker fexofenadine (5 – 10 μM) blocked these contractions (P<0.05; N=5), whereas the H2 receptor blocker cimetidine (5 – 10 μM) had no effect. In addition, transient receptor potential vanilloid 1 (TRPV1) channel blocker capsazepine significantly reduced histamine‐induced contractions only in bladder strips without urothelium, whereas the Na+ channel blocker tetrodotoxin (TTX) had no effect in any tissue (P < 0.05; N= 3–5). The mast cell activator compound 48/80 (10 μg/ml) contracted urinary bladder smooth muscle, and this contraction was also unaffected by TTX. These results indicate that H1 receptors on urinary bladder smooth cells likely modulate histamine‐induced bladder contraction, and sufficient amounts of histamine can be released from mast cells to cause a contraction. Support or Funding Information This research is supported by NIH K01‐DK103840 (NRT) and NIH R01‐DK119615 (NRT & GCM).