Bilateral versus ipsilateral neck dissection in oral and oropharyngeal cancer with contralateral cN0 neck.

OBJECTIVE: Contralateral elective neck dissection (cEND) in oral and oropharyngeal squamous cell cancer (OC/OPC) is still a matter of debate. The current study analyzed the outcome in OC/OPC patients with/without cEND. METHODS: OC/OPC patients (n = 471) were diagnosed with contralateral N0 after CT/MRI-scan combined with neck ultrasound. Clinico-pathological features were analyzed using Chi-square/Fisher exact/Student's t test. Survival rates were calculated using Kaplan-Meier and log-rank test. Prognostic variables were evaluated by Cox regression. Primary/secondary endpoints were overall/recurrence-free survival (OS/RFS). RESULTS: Pre-therapeutic imaging revealed a significantly over-staged N-status (p = 0.01), while occult contra-lateral N + was diagnosed in one patient only (0.4%). OC patients did not show differences in OS/RFS between the groups (ipsi- vs. bi-lateral). There was a strong tendency towards a better OS in OPC patients who underwent ipsi-lateral ND (p = 0.07). Cox-regression demonstrated that only tumor recurrence was associated with a fivefold increased risk of recurrence-associated death (p < 0.0001) that referred to a significant higher recurrence rate at primary tumor site (rT +) and increased distant metastatic outgrowth in OPC who underwent bi-lateral neck dissection (p = 0.03). While RFS of any cause (rT + /rN + /rM +) was significantly better in OPC with ipsi-lateral ND (p < 0.05), RFS of contralateral lymph node recurrence (rN2c) was comparable in both groups. CONCLUSION: END of the contralateral cN0 neck is not correlated by an increased RFS or OS. Standard imaging techniques including CT/MRI scan and neck ultrasound warrant watchful waiting for neck dissection of the contralateral cN0 neck.

Pharyngotomy in head and neck squamous cell carcinoma: functional and oncological aspects.

BACKGROUND: Head and Neck Surgery constantly has to oppose non-invasive organ preservation methods and therefore should be evaluated especially with regard to clinical and functional outcome. We will discuss the role of pharyngotomy in the treatment of HNSCC. METHODS: Seventy-three patients with carcinoma of the oral cavity, oro-/hypopharynx and supraglottis underwent lateral/median pharyngotomy. Functional and oncological parameters were retrospectively assessed and set into clinical context. RESULTS: The 5-year recurrence-free-interval (RFI) was significantly higher with surgery and adjuvant radio(chemo)therapy (80%; mean RFI: 92 months) when compared to conservative treatment (68%; mean RFI: 68 months). The 5-year overall-survival (OS) after surgery and conservative treatment was 71% and 54%, respectively. Compared to other surgical techniques (mean RFI: 82 months), pharyngotomy demonstrated a significant higher 5-year RFI (mean RFI: 89 months). CONCLUSIONS: Pharyngotomy achieves good exposure and clear resection margins that result in a notably good oncological outcome with a minimum of functional loss. In particular, among UICC IV oropharyngeal HNSCC, pharyngotomy is superior in OS and RFI to conservative methods.

In vitro and in vivo pharmacokinetic study of a dexamethasone-releasing silicone for cochlear implants.

Cochlear implants have been widely used for patients with profound hearing loss and partial deafness. Residual low-frequency hearing, however, may deteriorate due to insertion trauma and tissue response around the electrode array. The present study investigated in vitro and in vivo release of dexamethasone from silicone used for cochlear implant electrode carriers. The in vitro experiment involved an apparatus simulating the inner ear fluid environment in humans. Release from two sizes of silicone films (200 µm × 1 mm × 10 mm and 500 µm × 1 mm × 10 mm), each loaded with 2 % dexamethasone, and was measured for 24 weeks. In the in vivo experiment, silicone rods loaded with 2 or 10 % dexamethasone, respectively, were implanted into the scala tympani of guinea pigs. Perilymph concentrations were measured during the first week after implantation. The results showed that dexamethasone was released from the silicone in a sustained manner. After a burst release, perilymph concentration was similar for silicone incorporated with 2 and 10 % dexamethasone, respectively. The similar pharmacokinetic profile was found in the in vitro experiment. The period of sustained drug delivery was maintained for 20 weeks in vitro and for 1 week in vivo. The results of the present study suggest that drugs like dexamethasone are released in a controlled manner from silicon electrode carriers of cochlear implants. Further studies will identify optimal release profiles for the use with cochlear implants to improve their safety and long-term performance.

Outcome after pharyngeal reconstruction using pectoralis major and radial forearm flap after resection of pharyngeal and laryngeal squamous cell carcinomas.

The objective of this study is to assess the outcome after pharyngeal reconstruction using pectoralis major and radial forearm flaps in pharyngeal and laryngeal carcinomas. 90 patients who underwent flap surgery due to oro/-hypopharyngeal and laryngeal carcinomas were compared with 404 patients without pharyngeal reconstruction. Differences between the groups were analyzed using the Chi-square, Fisher exact, and the unpaired student's t test. Survival rates were calculated by Kaplan-Meier. Overall survival in oropharyngeal and hypopharyngeal/laryngeal cancer showed comparable results in patients with or without pharyngeal reconstruction (5-year: 53.4 vs. 64.2 %, p = 0.23; 5-year: 51.8 vs. 62.4 %, p = 0.94), while the survival time after flap surgery was significantly decreased (5-year: 44.8 vs. 62.4 %, p < 0.02; 5-year: 30.3 vs. 64.2 %, p = 0.07). Subgroup analysis attributed the worse survival after flap surgery to patients who underwent flap surgery due to functional deficits or recurrent disease (p = 0.002). In these patients, the median survival after flap surgery was 26 (hypopharyngeal/laryngeal cancer) or 13 months (oropharyngeal cancer) and associated with a significant increase in severe complications and hospitalization time (p < 0.0001). The hospitalization time correlated with the history of prior radiotherapy and the extent of surgery (r = 0.26; r = 0.3; p < 0.0001). Flap surgery in primary oropharyngeal and hypopharyngeal/laryngeal cancer showed an unaltered overall survival when compared with patients without reconstruction. Patients with recurrent disease or functional deficits demonstrated a significant decrease in survival combined with an increase of severe complications.

Liposomal treatment of xerostomia, odor, and taste abnormalities in patients with head and neck cancer.

BACKGROUND: Smell and taste disorders, sicca symptoms, can be detected in patients with head and neck cancer. The purpose of this study was to assess the utility of local liposomal application in the treatment of patients with head and neck cancers. METHODS: Ninety-eight patients with head and neck cancer were included in this study. The groups were defined as: group 1 = only surgery; group 2 = surgery + adjuvant radiochemotherapy; and group 3 = primarily radiochemotherapy. All patients had finished cancer treatment and received liposomal sprays for the nose and mouth for 2 months (LipoNasal, LipoSaliva; Optima Pharmaceutical GmbH, Germany) and suffered from taste and smell disorders. We performed tests with "Sniffin' Sticks," "Taste Strips," and a xerostomia questionnaire before and after treatment. RESULTS: After application of liposomes, patients demonstrated a statistically significant increase in smell and taste, and reduced xerostomia. CONCLUSION: Our results demonstrate that using nonpharmaceutical liposomal sprays improve smell, taste, and symptoms of xerostomia in patients with head and neck cancer. © 2015 Wiley Periodicals, Inc. Head Neck 38: E1232-E1237, 2016.

Effects of a dexamethasone-releasing implant on cochleae: A functional, morphological and pharmacokinetic study.

AIM: This study evaluated the impact of a dexamethasone-releasing silicone implant on hearing function preservation, cochlear morphology and perilymph pharmacokinetics after cochlear implantation. METHODS: Guinea pigs were implanted unilaterally with silicone rods containing either 2% dexamethasone (DEXA group, n = 18) or no dexamethasone (control group, n = 17). Auditory brainstem response (ABR) and distortion product otoacoustic emissions (DPOAEs) were measured preoperatively and over 6 months postoperatively. Cochlear histology using standard hematoxylin and eosin (H&E) staining and tumor necrosis factor (TNF)-alpha staining was performed 1 month postoperatively. Twenty-two guinea pigs were involved in the pharmacokinetic study, and real-time drug concentrations in perilymph were investigated using high-performance liquid chromatography (HPLC). The Mann-Whitney U test (1-tailed) was used for statistical analyses. RESULTS: ABR and DPOAE testing demonstrated decreased hearing function immediately postoperatively followed by a progressive hearing loss within the first day postoperatively. There was almost no observable hearing improvement in the control group from 1 week to 6 months postoperatively, but hearing levels in the DEXA group improved gradually from 1 week to 12 weeks. Hearing loss in the DEXA and control group was 5.0 ± 3.4 dB and 21.7 ± 5.3 dB, respectively at a 16-kHz stimulus frequency 6 months postoperatively. The difference in threshold shifts was present throughout all measured frequencies, and it was significant at 4-24 kHz. The morphological study revealed new fibrosis formation in the scala tympani, which encapsulated the implanted electrode. TNF-alpha positive staining in the cochleae of the DEXA group was less evident than the control group. The pharmacokinetic study revealed a peak perilymph concentration 30 min postoperatively and sustained dexamethasone release at least 1 week postoperatively. CONCLUSION: Cochlear implants that incorporate dexamethasone can release drug chronically in the inner ear and induce significant long-term recovery and preservation of auditory function after implantation.

Immunolocalization of antimicrobial and cytoskeletal components in the serous glands of human sinonasal mucosa.

Secretory cells in the seromucous glands of paranasal sinuses secrete antibacterial proteins for innate immune mucosal integrity. We studied the localization of antimicrobial and cytoskeletal components of the human seromucous glands and respiratory epithelium of the maxillary sinus and the ethmoidal cells by immunohistochemical methods. The presence of a variety of defense proteins such as lysozyme, lactoferrin, cathelicidin, and defensin-1, -2, -3 point to a crucial role in the immune defense for the respiratory tract. Cytoskeletal proteins such as actin, myosin 2, cytokeratin 7 and 19, α- and ß-tubulin, investigated for the first time in glands of paranasal sinuses, showed a stronger expression at the apical and lateral cell membrane. The localization of the cytoskeletal proteins might point to their participation in exocrine secretory processes and stabilizing effects.

Microvascular dysfunction in the course of metabolic syndrome induced by high-fat diet.

BACKGROUND: Metabolic syndrome (MetS) is associated with increased risk of cardiovascular disease (CVD). One important feature underlying the pathophysiology of many types of CVD is microvascular dysfunction. Although components of MetS are themselves CVD risk factors, the risk is increased when the syndrome is considered as one entity. We aimed to characterize microvascular function and some of its influencing factors in the course of MetS development. METHODS: Development of MetS in C57BL/6 mice on a high-fat diet (HFD, 51% of energy from fat) was studied. The initial phase of MetS (I-MetS) was defined as the first 2 weeks of HFD feeding, with the fully developed phase occurring after 8 weeks of HFD. We characterized these phases by assessing changes in adiposity, blood pressure, and microvascular function. All data are presented as mean ± standard error (SEM). Differences between cumulative dose-response curves of myograph experiments were calculated using non-linear regression analysis. In other experiments, comparisons between two groups were made with Student's t-test. Comparisons between more than two groups were made using one-way ANOVA with Tukey post-hoc test. A probability value <0.05 was considered statistically significant. RESULTS: I-MetS mice presented with weight gain, blood pressure elevation, and microvascular dysfunction characterized by augmented vasoconstriction. This finding, contrary to those in mice with fully developed MetS, was not associated with endothelial dysfunction, insulin resistance, or systemic inflammation. In the initial phase, perivascular adipose tissue showed no sign of inflammation and had no influence on the pattern of vasoconstriction. These findings suggest that the onset of hypertension in MetS is strongly influenced by vascular smooth muscle cell dysfunction and independent of important factors known to influence microvascular function and consequently blood pressure levels. CONCLUSION: We identified in I-MetS the occurrence of isolated augmented vasoconstriction along with blood pressure elevation, but not the presence of classical MetS components known to influence microvascular function. These findings increase our understanding of the pathophysiology of CVD risk associated with MetS.

Damage-associated molecular pattern activated Toll-like receptor 4 signalling modulates blood pressure in L-NAME-induced hypertension.

AIMS: Recent publications have shed new light on the role of the adaptive and innate immune system in the pathogenesis of hypertension. However, there are limited data whether receptors of the innate immune system may influence blood pressure. Toll-like receptor 4 (TLR4), a pattern recognition receptor, is a key component of the innate immune system, which is activated by exogenous and endogenous ligands. Hypertension is associated with end-organ damage and thus might lead to the release of damage-associated molecular patterns (DAMPs), which are endogenous activators of TLR4 receptors. The present study aimed to elucidate whether TLR4 signalling is able to modulate vascular contractility in an experimental model of hypertension thus contributing to blood pressure regulation. METHODS AND RESULTS: NG-nitro-l-arginine methyl ester (l-NAME)-induced hypertension was blunted in TLR4(-/-) when compared with wild-type mice. Treatment with l-NAME was associated with a release of DAMPs, leading to reactive oxygen species production of smooth muscle cells in a TLR4-dependent manner. As oxidative stress leads to an impaired function of the NO-sGC-cyclic GMP (cGMP) pathway, we were able to demonstrate that TLR4(-/-) was protected from sGC inactivation. Consequently, arterial contractility was reduced in TLR4(-/-). CONCLUSIONS: Cell damage-associated TLR4 signalling might act as a direct mediator of vascular contractility providing a molecular link between inflammation and hypertension.

Head and neck salivary gland carcinomas--elective neck dissection, yes or no?

PURPOSE: Surgical resection and subsequent neck dissection (ND) in cases of clinically positive lymph nodes is an accepted primary treatment strategy for salivary gland carcinomas. Because of uncertainty in the extent of lymphogenic metastasis, the authors advocate a strategy of surgical resection and elective ND (END) for all patients. The authors evaluated their treatment by estimating the frequency of metastatic disease and identifying factors associated with an increased risk for metastatic disease. MATERIALS AND METHODS: A retrospective cohort study was implemented using patient data obtained from the university's interdisciplinary board for head and neck tumors. Data were screened for age, gender, tumor entity, localization, grade, and TNM Classification of Malignant Tumors (by UICC, International Union Against Cancer) status. Statistical analysis was performed to identify possible predictors of lymph node metastasis. Nodal status groups (N(+) and N0) were compared with respect to age by t tests; other comparisons involved χ(2) tests. RESULTS: Ninety-four patients (50% female, 50% male; mean age, 59.12 yr) were identified, of whom 87 had an indication for END. On postsurgical histopathologic examination, 34 (39%; 17 male, 17 female) were diagnosed with N(+). Statistical analysis for nodal status produced explorative P values (age, P = .001; gender, P = .792; anatomic region, P = .114; tumor entity, P = .854; tumor status, P = .263; grade, P = .000). CONCLUSION: All studied malignancies were capable of lymph node dissemination. Therefore, no reliable preoperative predictors for lymphogenic metastasis are currently identifiable. Because of difficulties in safely predicting lymphogenic metastasis and the high rate of N(+) results on postoperative examination, the authors strongly advise END for all patients with salivary gland carcinoma.

Simulation of a MR-PET protocol for staging of head-and-neck cancer including Dixon MR for attenuation correction.

PURPOSE: To simulate and optimize a MR protocol for squamous cell cancer of the head and neck (HNSCC) patients for potential future use in an integrated whole-body MR-PET scanner. MATERIALS AND METHODS: On a clinical 3T scanner, which is the basis for a recently introduced fully integrated whole-body MR-PET, 20 patients with untreated HNSCC routinely staged with 18F-FDG PET/CT underwent a dedicated MR protocol for the neck. Moreover, a whole-body Dixon MR-sequence was applied, which is used for attenuation correction on a recently introduced hybrid MR-PET scanner. In a subset of patients volume-interpolated-breathhold (VIBE) T1w-sequences for lungs and liver were added. Total imaging time was analyzed for both groups. The quality of the delineation of the primary tumor (scale 0-3) and the presence or absence of lymph node metastases (scale 1-5) was evaluated for CT, MR, PET/CT and a combination of MR and PET to ensure that the MR-PET fusion does not cause a loss of diagnostic capability. PET was used to identify distant metastases. The PET dataset for simulated MR/PET was based on a segmentation of the CT data into 4 classes according to the approach of the Dixon MR-sequence for MR-PET. Standard of reference was histopathology in 19 cases. In one case no histopathological confirmation of a primary tumor could be achieved. RESULTS: Mean imaging time was 35:17 min (range: 31:08-42:42 min) for the protocol including sequences for local staging and attenuation correction and 44:17 min (range: 35:44-54:58) for the extended protocol. Although not statistically significant a combination of MR and PET performed better in the delineation of the primary tumor (mean 2.20) compared to CT (mean 1.40), MR (1.95) and PET/CT (2.15) especially in patients with dental implants. PET/CT and combining MR and PET performed slightly better than CT and MR for the assessment of lymph node metastases. Two patients with distant metastases were only identified by PET. CONCLUSION: We established a potential MR-protocol to be used for HNSCC patients in a recently introduced MR-PET scanner. The proposed protocol can be performed in an acceptable time frame and did not lead to a loss of diagnostic capability compared to PET/CT.

The human submandibular gland: immunohistochemical analysis of SNAREs and cytoskeletal proteins.

Submandibular acinar glands secrete numerous proteins such as digestive enzymes and defense proteins on the basis of the exocrine secretion mode. Exocytosis is a complex process, including a soluble NSF attachment protein receptor (SNARE)-mediated membrane fusion of vesicles and target membrane and the additional activation of cytoskeletal proteins. Relevant data are available predominantly for animal salivary glands, especially of the rat parotid acinar cells. The authors investigated the secretory molecular machinery of acinar (serous) cells in the human submandibular gland by immunohistochemistry and immunofluorescence and found diverse proteins associated with exocytosis for the first time. SNAP-23, syntaxin-2, syntaxin-4, and VAMP-2 were localized at the luminal plasma membrane; syntaxin-2 and septin-2 were expressed in vesicles in the cytoplasm. Double staining of syntaxin-2 and septin-2 revealed a colocalization on the same vesicles. Lactoferrin and α-amylase served as a marker for secretory vesicles and were labeled positively together with syntaxin-2 and septin-2 in double-staining procedures. Cytoskeletal components such as actin, myosin II, cofilin, and profilin are concentrated at the apical plasma membrane of acinar submandibular glands. These observations complement the understanding of the complex exocytosis mechanisms.

Inhibition of radiation induced migration of human head and neck squamous cell carcinoma cells by blocking of EGF receptor pathways.

BACKGROUND: Recently it has been shown that radiation induces migration of glioma cells and facilitates a further spread of tumor cells locally and systemically. The aim of this study was to evaluate whether radiotherapy induces migration in head and neck squamous cell carcinoma (HNSCC). A further aim was to investigate the effects of blocking the epidermal growth factor receptor (EGFR) and its downstream pathways (Raf/MEK/ERK, PI3K/Akt) on tumor cell migration in vitro. METHODS: Migration of tumor cells was assessed via a wound healing assay and proliferation by a MTT colorimeritric assay using 3 HNSCC cell lines (BHY, CAL-27, HN). The cells were treated with increasing doses of irradiation (2 Gy, 5 Gy, 8 Gy) in the presence or absence of EGF, EGFR-antagonist (AG1478) or inhibitors of the downstream pathways PI3K (LY294002), mTOR (rapamycin) and MEK1 (PD98059). Biochemical activation of EGFR and the downstream markers Akt and ERK were examined by Western blot analysis. RESULTS: In absence of stimulation or inhibition, increasing doses of irradiation induced a dose-dependent enhancement of migrating cells (p < 0.05 for the 3 HNSCC cell lines) and a decrease of cell proliferation (p < 0.05 for the 3 HNSCC cell lines). The inhibition of EGFR or the downstream pathways reduced cell migration significantly (almost all p < 0.05 for the 3 HNSCC cell lines). Stimulation of HNSCC cells with EGF caused a significant increase in migration (p < 0.05 for the 3 HNSCC cell lines). After irradiation alone a pronounced activation of EGFR was observed by Western blot analysis. CONCLUSION: Our results demonstrate that the EGFR is involved in radiation induced migration of HNSCC cells. Therefore EGFR or the downstream pathways might be a target for the treatment of HNSCC to improve the efficacy of radiotherapy.

Calcium sparks in the intact gerbil spiral modiolar artery.

BACKGROUND: Calcium sparks are ryanodine receptor mediated transient calcium signals that have been shown to hyperpolarize the membrane potential by activating large conductance calcium activated potassium (BK) channels in vascular smooth muscle cells. Along with voltage-dependent calcium channels, they form a signaling unit that has a vasodilatory influence on vascular diameter and regulation of myogenic tone. The existence and role of calcium sparks has hitherto been unexplored in the spiral modiolar artery, the end artery that controls blood flow to the cochlea. The goal of the present study was to determine the presence and properties of calcium sparks in the intact gerbil spiral modiolar artery. RESULTS: Calcium sparks were recorded from smooth muscle cells of intact arteries loaded with fluo-4 AM. Calcium sparks occurred with a frequency of 2.6 Hz, a rise time of 17 ms and a time to half-decay of 20 ms. Ryanodine reduced spark frequency within 3 min from 2.6 to 0.6 Hz. Caffeine (1 mM) increased spark frequency from 2.3 to 3.3 Hz and prolonged rise and half-decay times from 17 to 19 ms and from 20 to 23 ms, respectively. Elevation of potassium (3.6 to 37.5 mM), presumably via depolarization, increased spark frequency from 2.4 to 3.2 Hz. Neither ryanodine nor depolarization changed rise or decay times. CONCLUSIONS: This is the first characterization of calcium sparks in smooth muscle cells of the spiral modiolar artery. The results suggest that calcium sparks may regulate the diameter of the spiral modiolar artery and cochlear blood flow.

Tumor necrosis factor-α enhances microvascular tone and reduces blood flow in the cochlea via enhanced sphingosine-1-phosphate signaling.

BACKGROUND AND PURPOSE: We sought to demonstrate that tumor necrosis factor (TNF)-α, via sphingosine-1-phosphate signaling, has the potential to alter cochlear blood flow and thus, cause ischemic hearing loss. METHODS: We performed intravital fluorescence microscopy to measure blood flow and capillary diameter in anesthetized guinea pigs. To measure capillary diameter ex vivo, capillary beds from the gerbil spiral ligament were isolated from the cochlear lateral wall and maintained in an organ bath. Isolated gerbil spiral modiolar arteries, maintained and transfected in organ culture, were used to measure calcium sensitivity (calcium-tone relationship). In a clinical study, a total of 12 adult patients presenting with typical symptoms of sudden hearing loss who were not responsive or only partially responsive to prednisolone treatment were identified and selected for etanercept treatment. Etanercept (25 mg s.c.) was self-administered twice a week for 12 weeks. RESULTS: TNF-α induced a proconstrictive state throughout the cochlear microvasculature, which reduced capillary diameter and cochlear blood flow in vivo. In vitro isolated preparations of the spiral modiolar artery and spiral ligament capillaries confirmed these observations. Antagonizing sphingosine-1-phosphate receptor 2 subtype signaling (by 1 µmol/L JTE013) attenuated the effects of TNF-α in all models. TNF-α activated sphingosine kinase 1 (Sk1) and induced its translocation to the smooth muscle cell membrane. Expression of a dominant-negative Sk1 mutant (Sk1(G82D)) eliminated both baseline spiral modiolar artery calcium sensitivity and TNF-α effects, whereas a nonphosphorylatable Sk1 mutant (Sk1(S225A)) blocked the effects of TNF-α only. A small group of etanercept-treated, hearing loss patients recovered according to a 1-phase exponential decay (half-life=1.56 ± 0.20 weeks), which matched the kinetics predicted for a vascular origin. CONCLUSIONS: TNF-α indeed reduces cochlear blood flow via activation of vascular sphingosine-1-phosphate signaling. This integrates hearing loss into the family of ischemic microvascular pathologies, with implications for risk stratification, diagnosis, and treatment.

Sphingosine-1-phosphate-dependent activation of p38 MAPK maintains elevated peripheral resistance in heart failure through increased myogenic vasoconstriction.

RATIONALE: Mechanisms underlying vasomotor abnormalities and increased peripheral resistance exacerbating heart failure (HF) are poorly understood. OBJECTIVE: To explore the role and molecular basis of myogenic responses in HF. METHODS AND RESULTS: 10 weeks old C57Bl6 mice underwent experimental myocardial infarction (MI) or sham surgery. At 1 to 12 weeks postoperative, mice underwent hemodynamic studies, mesenteric, cerebral, and cremaster artery perfusion myography and Western blot. Organ weights and hemodynamics confirmed HF and increased peripheral resistance after MI. Myogenic responses, ie, pressure-induced vasoconstriction, were increased as early as 1 week after MI and remained elevated. Vasoconstrictor responses to phenylephrine were decreased 1 week after MI, but not at 2 to 6 weeks after MI, whereas those to endothelin (ET)-1 and sphingosine-1-phosphate (S1P) were increased at all time points after MI. An antagonist (JTE-013) for the most abundant S1P receptor detected in mesenteric arteries (S1P(2)R) abolished the enhanced myogenic responses of HF, with significantly less effect on controls. Mice with genetic absence of sphingosine-kinases or S1P(2)R (Sphk1(-/-); Sphk1(-/-)/Sphk2(+/-); S1P(2)R(-/-)) did not manifest enhanced myogenic responses after MI. Mesenteric arteries from HF mice exhibited increased phosphorylation of myosin light chain, with deactivation of its phosphatase (MLCP). Among known S1P-responsive regulators of MLCP, GTP-Rho levels were unexpectedly reduced in HF, whereas levels of activated p38 MAPK and ERK1/2 (extracellular signal-regulated kinase 1/2) were increased. Inhibiting p38 MAPK abolished the myogenic responses of animals with HF, with little effect on controls. CONCLUSIONS: Rho-independent p38 MAPK-mediated deactivation of MLCP underlies S1P/S1P(2)R-regulated increases in myogenic vasoconstriction observed in HF. Therapeutic targeting of these findings in HF models deserves study.

Sphingosine-1-phosphate modulates spiral modiolar artery tone: A potential role in vascular-based inner ear pathologies?

OBJECTIVE: The mechanisms regulating spiral modiolar artery (SMA) tone are not known, yet their characterization is pivotal for understanding inner ear blood flow regulation. Sphingosine-1-phosphate (S1P), known to stimulate vasoconstriction in several vascular beds, is a candidate regulator of SMA tone with potential pathophysiological relevance. METHODS: Gerbil SMAs were isolated, cannulated and pressurized (30 mm Hg transmural) for experimentation under near-in vivo conditions. For functional experiments, vascular diameter and intracellular Ca2+ were simultaneously measured. Standard RT-PCR and immunohistochemical techniques were also employed. RESULTS: mRNA transcripts encoding sphingosine kinase, S1P phosphohydrolase and three S1P receptors (S1P(1-3)) were detected in the SMA. S1P induced dose-dependent vasoconstriction of the SMA (EC50 = 115 nmol/L), and enhanced the apparent Ca2+-sensitivity of the contractile apparatus. Noradrenaline did not elicit vasoconstriction. The Rho kinase inhibitor Y27632 (1 micromol/L) reversed S1P-induced vasoconstriction and the S1P-mediated enhancement of Ca2+-sensitivity. RhoA was observed to translocate to the plasma membrane in response to stimulation with 30 micromol/L S1P. CONCLUSION: We conclude that all key signalling pathway constituents are present at the mRNA level for S1P to act as an endogenous regulator of SMA tone. S1P stimulates potent, RhoA/Rho kinase-dependent SMA vasoconstriction and Ca2+ sensitization. The high sensitivity to S1P suggests that SMA vasoconstriction is likely to occur under pathological conditions that increase intramural S1P concentrations (i.e., inflammation). From a clinical perspective, the present study identifies new potential therapeutic targets for the treatment of vascular-based, "stroke-like" inner ear pathologies: the enzymes responsible for S1P bioavailability and the S1P receptors.

Pharmacological reversal of endothelin-1 mediated constriction of the spiral modiolar artery: a potential new treatment for sudden sensorineural hearing loss.

BACKGROUND: Vasospasm of the spiral modiolar artery (SMA) may cause ischemic stroke of the inner ear. Endothelin-1 (ET-1) induces a strong, long-lasting constriction of the SMA by increasing contractile apparatus Ca2+ sensitivity via Rho-kinase. We therefore tested several Rho-kinase inhibitors and a cell-permeable analogue of cAMP (dbcAMP) for their ability to reverse ET-1-induced constriction and Ca2+-sensitization. METHODS: The present study employed SMA isolated from gerbil temporal bones. Ca2+sensitivity was evaluated by correlating vascular diameter and smooth muscle cell [Ca2+]i, measured by fluo-4-microfluorometry and videomicroscopy. RESULTS: The Rho-kinase inhibitors Y-27632, fasudil, and hydroxy-fasudil reversed ET-1-induced vasoconstriction with an IC50 of 3, 15, and 111 micromol/L, respectively. DbcAMP stimulated a dose-dependent vasodilation (Ec50 = 1 mmol/L) and a reduction of [Ca2+]i (EC50 = 0.3 micromol/L) of ET-1-preconstricted vessels (1 nmol/L). Fasudil and dbcAMP both reversed the ET-1-induced increase in Ca2+ sensitivity. CONCLUSION: Rho-kinase inhibition and dbcAMP reversed ET-1-induced vasoconstriction and Ca2+-sensitization. Therefore, Rho-kinase inhibitors or cAMP modulators could possess promise as pharmacological tools for the treatment of ET-1-induced constriction, ischemic stroke and sudden hearing loss.

Apical membrane P2Y4 purinergic receptor controls K+ secretion by strial marginal cell epithelium.

BACKGROUND: It was previously shown that K+ secretion by strial marginal cell epithelium is under the control of G-protein coupled receptors of the P2Y family in the apical membrane. Receptor activation by uracil nucleotides (P2Y2, P2Y4 or P2Y6) leads to a decrease in the electrogenic K+ secretion. The present study was conducted to determine the subtype of the functional purinergic receptor in gerbil stria vascularis, to test if receptor activation leads to elevation of intracellular [Ca2+] and to test if the response to these receptors undergoes desensitization. RESULTS: The transepithelial short circuit current (Isc) represents electrogenic K+ secretion and was found to be decreased by uridine 5'-triphosphate (UTP), adenosine 5'-triphosphate (ATP) and diadenosine tetraphosphate (Ap4A) but not uridine 5'-diphosphate (UDP) at the apical membrane of marginal cells of the gerbil stria vascularis. The potencies of these agonists were consistent with rodent P2Y4 and P2Y2 but not P2Y6 receptors. Activation caused a biphasic increase in intracellular [Ca2+] that could be partially blocked by 2-aminoethoxy-diphenyl borate (2-APB), an inhibitor of the IP3 receptor and store-operated channels. Suramin (100 microM) did not inhibit the effect of UTP (1 microM). The ineffectiveness of suramin at the concentration used was consistent with P2Y4 but not P2Y2. Transcripts for both P2Y2 and P2Y4 were found in the stria vascularis. Sustained exposure to ATP or UTP for 15 min caused a depression of Isc that appeared to have two components but with apparently no chronic desensitization. CONCLUSION: The results support the conclusion that regulation of K+ secretion across strial marginal cell epithelium occurs by P2Y4 receptors at the apical membrane. The apparent lack of desensitization of the response is consistent with two processes: a rapid-onset phosphorylation of KCNE1 channel subunit and a slower-onset of regulation by depletion of plasma membrane PIP2.

Endothelin-1-induced vasospasms of spiral modiolar artery are mediated by rho-kinase-induced Ca(2+) sensitization of contractile apparatus and reversed by calcitonin gene-related Peptide.

BACKGROUND AND PURPOSE: Vasospasms of the spiral modiolar artery may cause an ischemic stroke of the inner ear that manifests itself by a sudden hearing loss. Previously we have shown that endothelin-1 (ET-1) induces vasospasms of the spiral modiolar artery. Here we tested the hypotheses that ET-1-induced vasospasms are (1) reversible by ET(A) receptor antagonists; (2) mediated by a Ca(2+) sensitization of the contractile apparatus via a Rho-kinase-induced inhibition of myosin light chain phosphatase; and (3) reversible by the vasodilator calcitonin gene-related peptide (CGRP). METHODS: The Ca(2+) sensitivity of the contractile apparatus was evaluated by correlation between the smooth muscle cell Ca(2+) concentration and the vascular diameter, which were measured by microfluorometry with the fluorescent dye fluo-4 and videomicroscopy, respectively. RESULTS: ET-1-induced vasospasms were prevented but not reversed by the ET(A) receptor antagonists BQ-123 and BMS-182874. The Ca(2+) sensitivity of the contractile apparatus was increased by ET-1 and by inhibition of myosin light chain phosphatase with calyculin A and was decreased by CGRP. ET-1-induced vasospasms and Ca(2+) sensitization were prevented and reversed by the Rho-kinase antagonist Y-27632 and by CGRP. CONCLUSIONS: ET-1 induces vasospasms of the spiral modiolar artery via ET(A) receptor-mediated activation of Rho-kinase, inhibition of myosin light chain phosphatase, and an increase in Ca(2+) sensitivity, which is reversed by CGRP. The observation that vasospasms were reversed by Y-27632 but not by BQ-123 or BMS-182874 suggests that Rho-kinase, rather than the ET(A) receptor, is the most promising pharmacological target for the treatment of ET-1-induced vasospasms, ischemic strokes, and sudden hearing loss.