Video head impulse test for the assessment of vestibular function in patients with idiopathic sudden sensorineural hearing loss without vertigo.

OBJECTIVE: Idiopathic sudden sensorineural hearing loss may be accompanied by dizziness without true vertigo. This study used the video head impulse test to evaluate vestibular function in idiopathic sudden sensorineural hearing loss patients who described experiencing dizziness and not true vertigo. METHODS: A prospective study was conducted of 30 consecutive patients diagnosed with idiopathic sudden sensorineural hearing loss with dizziness without true vertigo. A comparison of the video head impulse test results of the patients who complained of dizziness (symptomatic group) with a group of patients with idiopathic sudden sensorineural hearing loss and no dizziness (asymptomatic) was performed. RESULTS: Nine patients (30 per cent) were symptomatic. Two of those patients had abnormal video head impulse test findings. Seven patients in the asymptomatic group (7 out of 21, 33 per cent) presented with abnormal video head impulse test results. No significant difference in vestibular function between the two groups was detected by the video head impulse test. CONCLUSION: The site of insult in patients with idiopathic sudden sensorineural hearing loss without true vertigo is usually limited to the cochlea or the cochlear nerve.

Non-melanoma skin cancer diagnosis: a comparison between dermoscopic and smartphone images by unified visual and sonification deep learning algorithms.

PURPOSE: Non-melanoma skin cancer (NMSC) is the most frequent keratinocyte-origin skin tumor. It is confirmed that dermoscopy of NMSC confers a diagnostic advantage as compared to visual face-to-face assessment. COVID-19 restrictions diagnostics by telemedicine photos, which are analogous to visual inspection, displaced part of in-person visits. This study evaluated by a dual convolutional neural network (CNN) performance metrics in dermoscopic (DI) versus smartphone-captured images (SI) and tested if artificial intelligence narrows the proclaimed gap in diagnostic accuracy. METHODS: A CNN that receives a raw image and predicts malignancy, overlaid by a second independent CNN which processes a sonification (image-to-sound mapping) of the original image, were combined into a unified malignancy classifier. All images were histopathology-verified in a comparison between NMSC and benign skin lesions excised as suspected NMSCs. Study criteria outcomes were sensitivity and specificity for the unified output. RESULTS: Images acquired by DI (n = 132 NMSC, n = 33 benign) were compared to SI (n = 170 NMSC, n = 28 benign). DI and SI analysis metrics resulted in an area under the curve (AUC) of the receiver operator characteristic curve of 0.911 and 0.821, respectively. Accuracy was increased by DI (0.88; CI 81.9-92.4) as compared to SI (0.75; CI 68.1-80.6, p < 0.005). Sensitivity of DI was higher than SI (95.3%, CI 90.4-98.3 vs 75.3%, CI 68.1-81.6, p < 0.001), but not specificity (p = NS). CONCLUSION: Telemedicine use of smartphone images might result in a substantial decrease in diagnostic performance as compared to dermoscopy, which needs to be considered by both healthcare providers and patients.

Calculated versus measured pure tone bone conduction 3 kHz thresholds in sudden sensorineural hearing loss.

OBJECTIVE: To compare the measured bone conduction threshold at 3 kHz with the calculated threshold in newly diagnosed sudden sensorineural hearing loss. METHODS: A retrospective chart review was conducted of pure tone audiograms in confirmed sudden sensorineural hearing loss cases. RESULTS: Of 157 patients with sudden sensorineural hearing loss, 144 had idiopathic hearing loss, 8 had vestibular schwannoma and 5 had Ménière's disease. The r value for the correlation between the two methods of 3 kHz assessment for all patients was 0.887 (p < 0.001). The mean difference between the measured and calculated 3 kHz thresholds was 0.76 ± 7.96 dB, 0.4 ± 8.08 dB and 1.5 ± 1.8 dB in the sudden sensorineural hearing loss, idiopathic and Ménière's disease groups, respectively. The mean difference between the measured and calculated 3 kHz thresholds was significantly greater in the vestibular schwannoma group (6.86 ± 4.38 dB) than in the idiopathic group (p = 0.013). CONCLUSION: The 3 kHz frequency may encompass important audiometric information. A discrepancy between the measured and calculated bone conduction 3 kHz thresholds raises suspicion of an underlying vestibular schwannoma as an aetiology for sudden sensorineural hearing loss, and these thresholds should therefore be measured independently and routinely.

Outcome of surgery for chronic suppurative otitis media with resistant Pseudomonas aeruginosa.

OBJECTIVE: To assess the effectiveness of surgical treatment of active chronic suppurative otitis media (CSOM) with ciprofloxacin-resistant Pseudomonas aeruginosa (P. aeruginosa) that failed comprehensive local and systemic treatment. STUDY DESIGN: Retrospective case review. SETTING: Tertiary referral center. PATIENTS: Eleven patients with ciprofloxacin-resistant P. aeruginosa CSOM that remained active despite comprehensive local and systemic treatment. All patients were operated by a single surgeon between February 2016 and July 2019 INTERVENTION(S): Tympanoplasty alone was performed in seven cases and accompanied by mastoidectomy in the other four cases. MAIN OUTCOME MEASURE(S): Resolution of infection and tympanic graft take on otoscopy. The secondary outcome measure is hearing. RESULTS: Tympanic graft take was successful and the infectious process was resolved in 8 out of the 11 cases, yielding a success rate of 73%. The average follow-up was 20 months. No surgical complications occurred. CONCLUSIONS: Tympanoplasty, with or without mastoidectomy, is safe and yields acceptable anatomical and functional success rates when intensive local and systemic treatment fails to stop the purulent discharge.

Increased incidence of Susac syndrome: a case series study.

BACKGROUND: Susac syndrome (SuS) is a rare condition characterized by a clinical triad of sensorineural hearing loss, branch artery occlusion and encephalopathy. This study reports an increased incidence of SuS in Israel. We describe the clinical characteristics of these patients, diagnostic procedures and the use and subsequent outcomes of newly published treatment guidelines. METHODS: This is a single center retrospective study. Patients who were diagnosed with SuS between July 2017 and August 2018 were enrolled in this study. RESULTS: Seven patients were diagnosed with SuS according to the diagnostic criteria in a time period of 13 months. The annual incidence was recently evaluated in Austria to be 0.024/100000, therefore, our case series represent at least a 5.4- fold increase in the annual incidence of SuS expected in Israel and a 7-fold increase in the annual incidence expected in our medical center. Mean time from the onset of the symptoms to diagnosis was three weeks and follow-up time was twenty four months. Recent exposure to cytomegalovirus was serologically evident in three patients and one patient had high titer of anti-streptolysin antibody. All patients underwent brain MRI, fluorescein angiography and audiometry. All patients were treated according to the newly recommended guidelines. All patients achieved clinical and radiological stability. CONCLUSIONS: We report of an increased incidence of SuS in Israel. Infectious serological findings may imply a post infectious mechanism. The use of the recommended diagnostic procedures reduced the time to diagnosis. Newly published treatment guidelines led to favorable clinical outcomes.

Eustachian tube dysfunction in candidates for surgery for obstructive sleep apnoea syndrome.

BACKGROUND: Defining the risk factors for Eustachian tube dysfunction can facilitate its prevention. It is hypothesised that Eustachian tube dysfunction as measured by the Eustachian Tube Dysfunction Questionnaire-7 is associated with obstructive sleep apnoea syndrome. METHODS: The questionnaire was systematically translated into Hebrew and validated in the accepted manner. This questionnaire was applied to obstructive sleep apnoea syndrome patients before and after expansion sphincter pharyngoplasty, in pre-set time intervals. The results were compared to those of controls from the general population. RESULTS: Thirty-one patients (males:females = 19:12) were enrolled in the obstructive sleep apnoea syndrome group. Mean age was 43 years (range, 31-55 years) and mean body mass index was 28 kg/m2 (range, 27-30 kg/m2). Median apnoea-hypopnea index (pre-operatively) was 34 events per hour. The questionnaire scores in expansion sphincter pharyngoplasty candidates were significantly worse than in controls (p < 0.001). Expansion sphincter pharyngoplasty did not change Eustachian tube function in the long term, but was associated with additional self-limiting Eustachian tube dysfunction in the first two post-operative months. CONCLUSION: Eustachian tube dysfunction is significantly worse in patients with obstructive sleep apnoea syndrome compared to controls. Expansion sphincter pharyngoplasty is not associated with Eustachian tube function improvement.

Integrin-based therapy of pancreatic adenocarcinoma: current status and future perspectives.

Pancreatic ductal adenocarcinoma (PDAC), although tenth in cancer incidence, holds the dubious distinction of being the fifth cause of cancer deaths in the Western countries and possibly the deadliest malignancy. Inoperable PDAC is characterized by late diagnosis, extensive metastases, extremely poor response to chemotherapy and, consequently, poor patients' prognosis-6.7% 5-year survival. PDAC reflects the failure of the medical profession to significantly prolong patients' lives and modest expectations for future cure. PDAC is characterized by extensive desmoplastic reaction, resulting in approximately 50% of tumor's volume consisting of non-tumor cells and extracellular matrix (ECM) stroma. These properties imply an important role for cell-ECM interaction, making cell-matrix adhesion molecules, such as integrins, of special interest as possible candidate targets for future anti-PDAC therapies. This review will attempt to overview the status of studies dealing with the involvement of integrins in the unique aggressive character of PDAC, the current status of experimental cancer therapies targeted at integrins, and the possible application of these preliminary clinical experiments to future PDAC therapy. I will also try to delineate the reasons for the failures of PDAC therapies and make some modest suggestions that might improve the health scientific community approaches to this extremely difficult problem.

Monitoring the effect of mild ischemia with a built-in light-emitting diode contact lens electrode and a low-cost custom-made apparatus.

Electroretinography (ERG) is widely used in clinical work and research to assess the retinal function. We evaluated an easy to build ERG setup adapted for small animals comprising two contact lens electrodes with a built-in light-emitting diode and a custom-made amplification system. The system's sensitivity was tested by monitoring ERG in albino rat eyes subjected to mild ischemia. Flash ERG was recorded by two contact lens electrodes positioned on the rat's corneas and used alternately as test or reference. The a- and b-wave amplitudes, a-wave latency, b-wave implicit time and oscillatory potentials (OPs) were analyzed. Ischemia was achieved by elevating the intraocular pressure in the eye's anterior chamber. ERG was recorded on post-ischemia (PI) days -1, 1, 3 and 7. Morphological changes were analyzed on hematoxylin/eosin stained 5 µm sections of control 7d PI retinas. In control eyes, ERG exhibited a pattern similar to a standard recording. Retinas subjected to mild ischemia preserved ordered layered morphology, exhibiting approximately 30% loss of ganglion cells and no changes in gross morphology. By day 3 PI, ischemia caused an increase in the a-wave amplitude (from 34.9 ± 2.7 to 45.4 ± 4.3 µV), a decrease in the b-wave amplitude (from 248 ± 13 to 162 ± 8 µV), an increase in a-wave latency (from 11.1 ± 0.3 to 17.3 ± 1.4 ms) and b-wave implicit time (from 81.0 ± 1.6 to 90.0 ± 2.5 ms), and attenuation of OPs. The described setup proved sensitive and reliable for evaluating subtle changes in the retinal function in small animals.

Evidence for a tinnitus subgroup responsive to somatosensory based treatment modalities.

Studies have established that the somatosensory system of the upper cervical region and head can be intimately involved in tinnitus. Tinnitus can arise directly from a disorder of the head and upper neck through activation of the somatosensory system. "Somatic testing" (a series of strong muscle contractions of the head and neck) can (1) modulate the tinnitus percept of approximately 80% of people with ongoing tinnitus, and (2) elicit a sound percept in approximately 50% of people with no tinnitus. These somatic phenomena are equally prevalent among people with or without functioning cochlea. Likely neural pathways underlying both the induction and modulation of tinnitus have been revealed in animal studies. Because somatic influences are fundamental to the operation of the auditory system, in general, and to tinnitus, in particular, somatic testing should be incorporated into all evaluations of tinnitus (1) to improve understanding of the role of the somatosensory system in any individual and (2) to identify subgroups of tinnitus patients who may respond to a particular treatment modality (as has already been shown for the tinnitus associated with temporomandibular disorder). Our clinical experience and review of reports of treatment modalities directed toward the somatosensory system supports the hypothesis that these modalities can benefit individuals with symmetric hearing thresholds but asymmetric widely fluctuating tinnitus. Treatment modalities involving the somatosensory system should be re-assessed by targeting this tinnitus subgroup.

Strontium-induced rat egg activation.

Parthenogenetic agents that evoke cytosolic calcium concentration ([Ca2+]i) oscillations similar to those evoked by sperm, mimic fertilization more faithfully than agents that trigger a single [Ca2+]i transient. Strontium chloride (SrCl2) binds to and activates the Ca2+-binding site on the inositol 1,4,5-trisphosphate receptor and evokes [Ca2+]i oscillations. Although SrCl2 has been reported to activate mouse eggs, little is known regarding the pattern of the [Ca2+]i oscillations it evokes in rat eggs and their effect on the early events of egg activation: cortical granule exocytosis (CGE) and completion of meiosis (CM). In the current study we investigated the effect of various concentrations of SrCl2 (2, 4 or 6 mM) on [Ca2+]i, by monitoring [Ca2+]i oscillations in fura-2-loaded rat eggs. Treatment with 2 mM SrCl2 was optimal for inducing the first [Ca2+]i transient, which was similar in duration to that triggered by sperm. However, the frequency and duration of the subsequent [Ca2+]i oscillations were lower and longer in SrCl2-activated than in sperm-activated eggs. The degree of CGE was identical in eggs activated by either sperm or SrCl2, as assessed by semi-quantitative immunohistochemistry combined with confocal microscopy. Evoking 1, 2 or 10 [Ca2+]i oscillations (8, 15 or 60 min in SrCl2 respectively) had no effect on the intensity of fluorescent CGE reporter dyes, while 60-min exposure to SrCl2 caused a delay in CM. Our results demonstrate that SrCl2 is an effective parthenogenetic agent that mimics rat egg activation by sperm, as judged by the generation of [Ca2+]i oscillations, CGE and CM.

Genetic synthetic lethality screen at the single gene level in cultured human cells.

Recently, we demonstrated the feasibility of a chemical synthetic lethality screen in cultured human cells. We now demonstrate the principles for a genetic synthetic lethality screen. The technology employs both an immortalized human cell line deficient in the gene of interest, which is complemented by an episomal survival plasmid expressing the wild-type cDNA for the gene of interest, and the use of a novel GFP-based double-label fluorescence system. Dominant negative genetic suppressor elements (GSEs) are selected from an episomal library expressing short truncated sense and antisense cDNAs for a gene likely to be synthetic lethal with the gene of interest. Expression of these GSEs prevents spontaneous loss of the GFP-marked episomal survival plasmid, thus allowing FACS enrichment for cells retaining the survival plasmid (and the GSEs). The dominant negative nature of the GSEs was validated by the decreased resident enzymatic activity present in cells harboring the GSEs. Also, cells mutated in the gene of interest exhibit reduced survival upon GSE expression. The identification of synthetic lethal genes described here can shed light on functional genetic interactions between genes involved in normal cell metabolism and in disease.

Establishment of a chemical synthetic lethality screen in cultured human cells.

The synthetic lethality screen is a powerful genetic method for unraveling functional interactions between proteins in yeast. Here we demonstrate the feasibility of a chemical synthetic lethality screen in cultured human cells, based in part on the concept of the yeast method. The technology employs both an immortalized human cell line, deficient in a gene of interest, which is complemented by an episomal survival plasmid expressing the gene of interest, and the use of a novel double-label fluorescence system. Selective pressure imposed by any one of several synthetic lethal metabolic inhibitors prevented the spontaneous loss of the episomal survival plasmid. Retention or loss over time of this plasmid could be sensitively detected in a blind test, while cells were grown in microtiter plates. Application of this method should thus permit high throughput screening of drugs, which are synthetically lethal with any mutant human gene of interest, whose normal counterpart can be expressed. This usage is particularly attractive for the search of drugs, which kill malignant cells in a gene-specific manner, based on their predetermined cellular genotype. Moreover, by replacing the chemicals used in this example with a library of either DNA oligonucleotides or expressible dominant negative genetic elements, one should be able to identify synthetic lethal human genes.

Constitutive signaling by Kaposi's sarcoma-associated herpesvirus G-protein-coupled receptor desensitizes calcium mobilization by other receptors.

We coexpressed Kaposi's sarcoma-associated herpesvirus G protein-coupled receptors (KSHV-GPCRs) with thyrotropin-releasing hormone (TRH) receptors or m1-muscarinic-cholinergic receptors in Xenopus oocytes and in mammalian cells. In oocytes, KSHV-GPCR expression resulted in pronounced (81%) inhibition (heterologous desensitization) of Ca(2+)-activated chloride current responses to TRH and acetylcholine. Similar inhibitions of cytoplasmic free Ca(2+) responses to TRH were observed in human embryonic kidney HEK 293 EM cells and in mouse pituitary AtT20 cells. Further study of oocytes showed that this inhibition was partially reversed by interferon-gamma-inducible protein 10 (IP-10), an inverse agonist of KSHV-GPCR. The basal rate of (45)Ca(2+) efflux in oocytes expressing KSHV-GPCRs was 4.4 times greater than in control oocytes, and IP-10 rapidly inhibited increased (45)Ca(2+) efflux. In the absence of IP-10, growth-related oncogene alpha caused a further 2-fold increase in (45)Ca(2+) efflux. In KSHV-GPCR-expressing oocytes, responses to microinjected inositol 1,4,5-trisphosphate were inhibited by 74%, and this effect was partially reversed by interferon-gamma-inducible protein 10. Treatment with thapsigargin suggested that the pool of calcium available for mobilization by TRH was decreased in oocytes coexpressing KSHV-GPCRs. These results suggest that constitutive signaling by KSHV-GPCR causes heterologous desensitization of responses mediated by other receptors, which signal via the phosphoinositide/calcium pathway, which is caused by depletion of intracellular calcium pools.

A hyperosmotic stimulus elevates intracellular calcium and inhibits proliferation of a human keratinocyte cell line.

Occlusion has previously been used to treat psoriatic plaques and was shown to improve the condition. We investigated the consequences of applying a mechanical stress, in vitro, on the HaCaT keratinocyte cell line. A mechanical load applied to cells can be mimicked by a hyperosmotic stimulus. Exposure of HaCaT keratinocytes to different hyperosmotic solutions (final osmolarity in the range 350-600 mOsm, produced by sucrose addition) resulted in an inhibition of cell proliferation after 96 h of treatment. As keratinocyte maturation is regulated by calcium levels, we measured hyperosmotic-stimulus-induced changes of intracellular calcium ([Ca2+]i) by single-cell image analysis employing FURA-2/AM. The hyperosmotic stimulus produced a rapid transient 2.6-fold elevation of [Ca2+]i followed by a gradual decay to the basal level. The transients originated from extracellular as well as from intracellular calcium pools and did not respond to voltage-sensitive calcium channel blockers. The hyperosmotic stimulus was shown to increase the cellular expression of involucrin, a differentiation marker, following 72 h of incubation, as measured by flow cytometry. Treatment of cells with the [Ca2+]i chelator BAPTA/AM almost completely blocked the [Ca2+]i elevation, but did not alter cellular growth or the induction of differentiation observed after hyperosmotic stimulus. It is suggested that treatment of keratinocytes with hyperosmotic stimulus can induce short-time effects (calcium transients) as well as long-term cellular maturation.

[Hospitalization for renal colic in the emergency room of a large central hospital in Israel: epidemiological features and clinical manifestations].

The medical records of all patients referred to the emergency department (ED) of Sheba Medical Center for renal colic during 1996 were analyzed. Patients discharged from the ED and those hospitalized were compared. There was no significant difference between the 2 groups with regard to average age or sex distribution. Statistically significant differences were found with regard to frequency of chills and fever, history of renal colic, referral for renal colic during that year or hospitalization for renal colic or nephrolithiasis, previous positive imaging, stone removal by surgery or extracorporeal shock wave lithotripsy, fever exceeding 37.5 degrees and administration of fluids, pethidine or pramin in the ED, prolonged stay in the ED and previous appendectomy. A conditional regression model tested the predictive value of each of those factors. Inclusion of independent variables into the model led to an overall correct classification rate of 84.43%, with 44.83% sensitivity and 93.16% specificity. There were correlations between referrals for renal colic, overall renal colic rate and average monthly temperature, so there was no pure correlation between average monthly temperature and referrals to the ED for renal colic. The major indications for hospitalization were actually the clinical ones, indicating either an active metabolic disease or suspected obstruction of the urinary tract. Treatment in the ED and duration of the visit indicated disease severity.

Rapid desensitization of the TRH receptor and persistent desensitization of its constitutively active mutant.

We studied rapid desensitization of the thyrotropin-releasing hormone receptor (TRH-R) or the m1-muscarinic receptor (m1-R) to a short challenge of threshold TRH concentration and persistent desensitization due to constitutive activity of a mutant TRH-R. Xenopus oocytes expressing TRH-Rs and/or m1-Rs were challenged for 15 s with threshold concentrations of TRH ([TRH]) and then immediately with supraoptimal [TRH] or acetylcholine ([ACh]). The threshold challenge caused desensitization of 50 - 57% of responses to subsequent supraoptimal stimulation with TRH or ACh. The homologous desensitization was reversible within 60 s after removal of the agonist. The protein kinase C (PKC) inhibitor, chelerythrine, inhibited the control responses by 30 - 40%, without affecting the desensitized responses. Chelerythrine or the phosphatase inhibitor, okadaic acid, had little effect on the kinetics of resensitization, indicating limited involvement of PKC. In oocytes coexpressing wild type TRH-Rs or m1-Rs with a constitutively active TRH-R mutant (C335Stop TRH-R), a persistent desensitization (33 - 57%) of the responses to TRH or ACh was observed. Additionally, there was a complete loss of the rapid desensitization induced by threshold [TRH]. Chlorodiazepoxide (CDE), a competitive binding antagonist of TRH-Rs and an inverse agonist of C335Stop TRH-Rs, abolished the persistent desensitization induced by C335Stop TRH-Rs and enabled the rapid desensitization, conferring the wild type phenotype on C335Stop TRH-Rs. Chelerythrine had qualitatively the same effect as CDE. In conclusion, unlike the rapid desensitization, the persistent desensitization caused by the constitutively active C335Stop TRH-Rs is largely mediated by PKC. It abrogates, however, the rapid desensitization, suggesting a common mechanistic step(s).

Nonadrenergic, noncholinergic relaxation of bovine iris sphincter: role of endogenous nitric oxide.

PURPOSE: To investigate the role of endogenously generated nitric oxide (NO) in the relaxation of bovine iris sphincter. METHODS: Isolated bovine sphincters were mounted on an isometric tension apparatus. Contraction-relaxation response was elicited by electrical field stimulation (ES; 12 Hz, 50-msec duration, 70-80 V). Relaxation was arbitrarily defined as maximal decrease of tension below prestimulation baseline after cessation of ES. We also determined the tissue levels of cyclic guanosine monophosphate (cGMP) by radioimmunoassay. RESULTS: ES produced a biphasic response: contraction followed by relaxation. After cessation of ES, the muscle relaxed to below the initial baseline tension. Tetrodotoxin (TTX) abolished most of the contraction and all the relaxation response. Atropine blocked most of the contraction component, leaving the relaxation component unchanged. Prazosin and bupranolol (alpha1-adrenergic and beta-adrenergic antagonists, respectively) also did not affect the relaxation component of the response. Neither substance P nor its antagonist (N-acetyl-L-tryptophane 3,5-bis (trifluoromethyl)-benzyl ester; ATTB) inhibited or mimicked the response. The nitric oxide synthase (NOS) inhibitors Nomega-nitro-L-arginine methyl ester (L-NAME), Nomega-nitro-L-arginine (L-NNA), and aminoguanidine dose-dependently inhibited the relaxation response by 50% to 70%. The free radical scavenger 2-(4-carboxyphenyl) 4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (carboxy-PTIO) and the guanylyl cyclase inhibitor methylene blue also abrogated 70% and 45% of the relaxation response, respectively. ES caused an increase in muscle cGMP from 2.3+/-0.3 to 3.9+/-0.5 picomoles per muscle. L-NNA or L-NAME significantly decreased the tissue cGMP content (to 1.2+/-0.1 picomoles per muscle) and prevented the increase caused by ES. CONCLUSIONS: The relaxation component of the iris sphincter response to ES is a distinct nonadrenergic, noncholinergic, ES-induced event. Most of the relaxation is mediated by the endogenously generated NO-guanylyl cyclase-cGMP cascade.

The effect of divalent cations on bovine retinal NOS activity.

The divalent cation requirements of NOS activity in bovine retina homogenate supernatant were investigated. Supernatants were assayed under standard conditions (in mM: EDTA 0.45, Ca2+ 0.25, Mg2+ 4.0). In order to investigate the enzyme's dependence on divalent cations, the tissue homogenate was depleted of di- and trivalent cations by passing it over a cation-exchange column (Chelex 100). Surprisingly, NOS activity was 50-100% higher in this preparation. However, addition of either EDTA (33 microM) or EGTA (1 mM) almost fully inhibited NOS activity, suggesting a requirement for residual divalent metal cation(s). Phenanthroline or iminodiacetic acid at low concentrations had little effect on activity, suggesting no requirement for Fe2+, Zn2+ or Cu2+. Ca2+ had a moderate stimulatory effect, with an optimum activity around 0.01 mM. Mg2+ or Mn2+ had little effect at concentrations < 0.25 mM. However, in the presence of EDTA, Mn2+ or Ca2+ markedly stimulated NOS activity with the optimum at 0.1 mM. At high concentrations (> 0.1-0.2 mM), all divalent cations tested (Ba2+, Zn2+, Co2+, Mn2+, Mg2+, Ca2+), as well as La3+, dose-dependently inhibited NOS activity. We propose that retinal NOS requires low concentrations of naturally occurring divalent metal ions, most probably Ca2+, for optimal activity and is inhibited by high di- and trivalent metal concentrations, probably by competition with Ca2+.

Inverse agonist abolishes desensitization of a constitutively active mutant of thyrotropin-releasing hormone receptor: role of cellular calcium and protein kinase C.

1. C335Stop is a constitutively active mutant of the TRH receptor (TRH-R). To investigate the mechanism of the decreased responsiveness of C335Stop TRH-R, we studied cellular Ca2+ concentrations ([Ca2+]i) in AtT20 cells stably transfected with C335Stop TRH-R cDNA, or Ca2+-activated chloride currents in Xenopus laevis oocytes expressing this mutant receptor after injection of cRNA. The competitive TRH-R binding antagonist, chlorodiazepoxide (CDE), was used as an inverse agonist to study the contribution of constitutive activity to desensitization. 2. Acute treatment with CDE resulted in a rapid (within minutes) decrease in [Ca2+]i and an increase in the response amplitude to TRH with no measurable change in receptor density. Conversely, removal of chronically administered CDE caused a rapid increase in [Ca2+]i and a decrease in TRH response amplitude. 3. CDE abolished heterologous desensitization induced by C335Stop TRH-R on muscarinic m1-receptor (ml-R) co-expressed in Xenopus oocytes. 4. Chelation of extracellular calcium with EGTA caused a rapid decrease in [Ca2+]i and a concomitant increase in the response to TRH in AtT20 cells expressing C335Stop TRH-Rs. 5. Chelerythrine, a specific inhibitor of protein kinase C (PKC), reversed the heterologous desensitization of the response to acetylcholine (ACh). The phosphoserine/phosphothreonine phosphatase inhibitor, okadaic acid, abolished the effect of chelerythrine. 6. Down-regulation of PKC by chronic exposure to phorbol 12-myristate 13-acetate (PMA) or acute inhibition with chelerythrine caused a partial resensitization of the response to TRH. 7. Western analysis indicated that the alpha subtype of protein kinase C was down-regulated in cells expressing C335Stop TRH-Rs. Following a 5 min exposure to PMA, the residual alphaPKC translocated to the particular fraction. 8. We propose that cells expressing the constitutively active mutant TRH-R rapidly desensitize their response, utilizing a mechanism mediated by an increase in [Ca2+]i and PKC.