Primary total laryngectomy and pharyngolaryngectomy in T4 pharyngolaryngeal cancers: Oncologic and functional results and prognostic factors.

OBJECTIVES: The aims of this study were to assess oncologic and functional outcome in primary total laryngectomy or pharyngolaryngectomy (TL/TL/TPL) for laryngeal or hypopharyngeal cancer with extra-laryngeal extension (T4) and to determine the predictive factors of these results. MATERIAL AND METHODS: A retrospective analysis was performed on the computerized medical records of all patients undergoing primary TL/TPL for T4 larynx or hypopharynx squamous cell carcinoma between 2000 and 2014 at our institution. Predictive factors of oncologic and functional outcome were investigated on univariate and multivariate analysis. RESULTS: Sixty-three patients (58 men, 5 women; mean age, 68.8±9.7 years) were included. Overall and disease-specific survivals were 69% and 80% at 3 years, and 56% and 69% at 5 years, respectively. On multivariate analysis, gender (female, P<0.001), ASA score (ASA≥3; P=0.006) and vascular embolism (P=0.006) had significant pejorative impact on overall survival. Six months after end of treatment, 90% of patients had recovered independent oral feeding and 83% of those with tracheoesophageal voice prostheses had recovered an intelligible voice. CONCLUSION: Primary TL/TPL remains the gold standard treatment for T4 larynx or hypopharynx cancer. It provides satisfactory oncologic and functional outcomes.

Transoral-transcervical oropharyngectomy without mandibulotomy, associated to fasciocutaneous radial forearm free-flap reconstruction, for oropharyngeal cancer: Postoperative course, oncologic and functional results, and prognostic factors.

OBJECTIVE: To analyze postoperative course, oncologic and functional results and prognostic factors of transoral-transcervical oropharyngeal cancer surgery without mandibulotomy, associated to radial forearm free-flap reconstruction. MATERIAL AND METHODS: Retrospective analysis of computerized medical records of all patients who underwent this type of surgery in our institution between 2004 and 2014. Predictive factors of oncologic and functional results were investigated on univariate and multivariate analyses. RESULTS: Forty-four patients (37 male, 7 female; mean age, 62.3±9.3years) were included. Three-year overall, disease-specific and recurrence-free survival was 90%, 92% and 79%, respectively. Functional scores were satisfactory (normal or slight impairment) for feeding, speech and oral opening functions in 86%, 93% and 100% of cases, respectively. ASA score≥III had significantly negative impact on overall survival (P=0.005) and on feeding (P=0.01) and speech (P=0.01). CONCLUSION: Transoral-transcervical oropharyngeal cancer surgery without mandibulotomy provided excellent oncologic and functional outcomes; it is an advantageous alternative to the conventional conservative transmandibular oropharyngectomy.

Reduced infections with perioperative immunonutrition in head and neck cancer: exploratory results of a multicenter, prospective, randomized, double-blind study.

BACKGROUND & AIMS: Head and neck cancer surgery is affected by complications in 20-60% of cases, with risk factors being malnutrition, alcoholism and immunosuppression due to cancer. The aim of the study was to investigate whether preoperative or perioperative immunonutrition could reduce postoperative infectious complications (IC) and surgical-site infections (SSI) in this population. METHODS: This was a multicenter, prospective, randomized, double-blind study. Patients with oropharyngeal and pharyngolaryngeal tumour were randomly allocated to three groups: a) perioperative formula of Impact(®) without immune nutrients, named "reference diet" (group A, control); b) preoperative Impact(®) and "reference diet" postoperatively (group B); c) Impact(®) perioperatively (group C). Products were available in oral and enteral formula and were given 7 days before surgery and for 7-15 days postoperatively. The primary and secondary endpoints were the incidence of IC and SSI, respectively. RESULTS: Of 312 randomized patients, 205 were evaluable for ITT analysis. There was no significant difference in IC and SSI. However out of this population, only 64 patients had taken at least 75% of the theoretical intake from surgery to day 10 (per-protocol population). In this condition, a significant difference in IC (OR = 0.24, p = 0.05), SSI (OR = 0.17, p = 0.04) and also in the median length of postoperative stay (18 vs. 25 days, p = 0.05) was demonstrated between groups A and C. CONCLUSIONS: In the ITT population, no significant difference in IC, SSI and LOS was demonstrated. Positive exploratory results on the perioperative Impact(®) per-protocol population, encourage further study in head and neck cancer patients. Registered under ClinicalTrials.gov Identifier no. NCT00765440.

Impact of preoperative radiotherapy on head and neck free flap reconstruction: a report on 429 cases.

BACKGROUND: The primary objective of this study was to evaluate the impact of preoperative radiotherapy on the outcomes of head and neck microvascular reconstruction. The secondary objective was to assess the specific effects of irradiation doses (IDs) ≥60 Gy on the outcomes of head and neck microvascular reconstruction. METHODS: All patients who underwent head and neck free-flap reconstruction in our institution between 2000 and 2010 were included in this retrospective study. A total of 429 patients were enrolled including 136 patients previously irradiated on the head and neck. The impact of preoperative radiotherapy on free-flap success, local and general complications, postoperative mortality, time of decannulation, duration of enteral nutrition and length of stay was assessed in univariate and multivariate analyses. RESULTS: In multivariate analysis, preoperative radiotherapy (irrespective of ID) was a significant risk factor for fistula formation (p = 0.003) and wound infection (p = 0.005). Previous neck irradiation at doses ≥60 Gy was associated with an increased risk of free-flap failure (p = 0.04), overall local complications (p = 0.05), haematoma (p = 0.04) and longer duration of enteral nutrition (p = 0.006) and hospital stay (p = 0.004). CONCLUSIONS: Preoperative radiotherapy, particularly for ID ≥ 60 Gy, is one of the main determinants of the outcomes of head and neck microvascular reconstruction.

ATP-sensitive potassium channels (K(ATP)) in retina: a key role for delayed ischemic tolerance.

The objectives of the present study were to determine the localization of K(ATP) channels in normal retina and to evaluate their potential roles in ischemic preconditioning (IPC) in a rat model of ischemia induced by increased intraocular pressure (IOP). Brown Norway rats were subjected to sublethal 3-, lethal 20- and 40-min ischemia and the functional recovery was evaluated using electroretinography. The time interval between ischemic insults ranged from 1 to 72 h. The effects of K(ATP) channel blockade on IPC protection were studied by treatment with 0.01% glipizide. IPC was mimicked by injection of K(ATP) channel openers of 0.01% (-)cromakalim or 0.01% P1060 72 h before 20-min ischemia. Co-expression of K(ATP) channel subunits Kir6.2/SUR1 was observed in the retinal pigment epithelium, inner segments of photoreceptors, outer plexiform and ganglion cell layers and at the border of the inner nuclear layer. In contrast to a 20- or 40-min ischemia, a 3-min ischemia induced no alteration of the electroretinogram (ERG) and constituted the preconditioning stimulus. An ischemic challenge of 40 min in preconditioned rats induced impairment of retinal function. However, animals preconditioned 24, 48 and 72 h before 20-min ischemia had a significant improvement of the ERG. (-)Cromakalim and P1060 mimicked the effect of IPC. Glipizide significantly suppressed the protective effects of preconditioning. In conclusion, activation of K(ATP) channels plays an important role in the mechanism of preconditioning by enhancing the resistance of the retina against a severe ischemic insult.

In vivo cytotoxicity of the prion protein fragment 106-126.

Transmissible spongiform encephalopathies are fatal neurological diseases characterized by astroglyosis, neuronal loss, and by the accumulation of the abnormal isoform of the prion protein. The amyloid prion protein fragment 106-126 (P106-126) has been shown to be toxic in cultured hippocampal neurons (). Here, we show that P106-126 is also cytotoxic in vivo. Taking advantage of the fact that retina is an integral part of the central nervous system, the toxic effect of the peptide was investigated by direct intravitreous injection. Aged solutions of P106-126 induced apoptotic-mediated retinal cell death and irreversibly altered the electrical activity of the retina. Neither apoptosis nor electroretinogram damages were observed with freshly diluted P106-126, suggesting that the toxicity is linked to the aggregation state of the peptide. The retina provides a convenient in vivo system to look for potential inhibitors of cytotoxicity associated with spongiform encephalopathies.

Immunolocalization of the arachidonic acid and mechanosensitive baseline traak potassium channel in the nervous system.

TRAAK is the sole member of the emerging class of 2P domain K+ channels to be exclusively expressed in neuronal cells. TRAAK produces baseline K+ currents which are strongly stimulated by arachidonic acid and by mechanical stretch, and which are insensitive to the classical K+ channel blockers tetraethylammonium, Ba2+, and Cs+. This report describes the immunolocalization of TRAAK in brain, spinal cord, and retina of the adult mouse. The most striking finding is the widespread distribution of the TRAAK immunoreactivity, with a prominent staining of the cerebellar cortex, neocortex, hippocampus, dentate gyrus, subiculum, the dorsal hippocampal commissure, thalamus, caudate-putamen, olfactory bulb, and several nuclei in the brainstem. Virtually all neurons express TRAAK, and the highest immunoreactivity was seen in soma, and to a lesser degree in axons and/or dendrites in most areas in brain and spinal cord. In the retina, the TRAAK protein is concentrated to the soma of ganglion cells and to the dendrites of all other neurons. Taken together, these results show a wide distribution of TRAAK, a mechanosensitive and arachidonic acid-stimulated neuron-specific baseline K+ channel, in brain, spinal cord and retina.

Riluzole improves functional recovery after ischemia in the rat retina.

PURPOSE: Retinal ischemia leads to neuronal death. The effects of riluzole, a drug that protects against the deleterious effect of cerebral ischemia by acting on several types of ion channels and blocking glutamatergic neurotransmission, were investigated in a rat model of retinal ischemic injury. METHODS: Retinal ischemia was induced by increasing intraocular pressure above systolic blood pressure for 30 minutes. Electroretinograms were recorded before ischemia and at different periods of reperfusion. Riluzole was injected or topically applied to the eye before or after ischemia and twice daily during the reperfusion period. Retinas were harvested for histopathology (toluidine blue and silver-impregnation stainings, Tdt-dUTP terminal nick-end labeling [TUNEL] method) and immunohistochemistry for cytoskeletal glial fibrillary acid protein and c-jun NH2-terminal kinase (p-JNK). RESULTS: Ischemia for 30 minutes caused a reduction of a- and b-waves of the electroretinogram. Systemic and topical treatments with riluzole significantly enhanced the recovery of the reduced a- and b-waves after defined reperfusion times. Riluzole also prevented or attenuated ischemia-induced retinal cell death (necrosis and apoptosis) and reduced the activation of p-JNK, c-jun phosphorylation, and the increase of cytoskeletal proteins induced by ischemic injury. CONCLUSIONS: Riluzole acted in vivo as a potent neuroprotective agent against pressure-induced ischemia. Therefore, riluzole may be a major drug for use in protection against retinal injury.

Effects of EGb761 and superoxide dismutase in an experimental model of retinopathy generated by intravitreal production of superoxide anion radical.

BACKGROUND: A study was carried out to investigate the effect of two antioxidants--Ginkgo biloba extract (EGb761) and superoxide dismutase (SOD)--in an experimental model of vitreoretinopathy obtained by direct production of oxygen free radicals in the vitreous cavity. METHODS: Twenty-eight pigmented rabbits were used. Vitreoretinopathy was induced by intravitreal injection of 50 microliters of a mixture composed of 40 nmol of xanthine and 0.001 IU of xanthine oxidase. Rabbits were randomly distributed into four groups: Group 1 (n = 8) did not receive any treatment and served as a positive control. Groups 2 (n = 8) and 3 (n = 8) received for 1 month EGb761 given orally at a dose of 100 mg/kg/day, respectively 1 day after and 1 week before induction of retinopathy. Group 4 (n = 4) was treated by three intramuscular injections of 15,000 IU/kg of SOD, 24 h before induction and 24 and 48 h thereafter. Clinical evaluations and electroretinograms (ERG) were repeatedly performed until the animals were killed at day 28. Histological examinations and immunohistological procedures were performed to ascertain the origin and characteristics of the cellular proliferation and to compare vitreoretinal structures in the four groups. RESULTS: Intravitreal injection of xanthine-xanthine oxidase produced a strong inflammatory response with vitreous infiltrates and epiretinal membrane formation, inconstantly associated with retinal detachment. ERG showed a decrease of the a-, b- and c-waves beginning within a few hours after injection. Histologic evaluation found an intravitreal and epiretinal infiltration by leukocytes and epithelial-derived cells, dense vitreoretinal membranes and retinal detachments with occasional neovascularization. In the treated groups (groups 2-4), all clinical, electric and histologic data were significantly improved compared to the control group. However, no difference could be found among the three treated groups. CONCLUSION: This study demonstrates the strong pathologic effects of free radical production on the retina and the close relationships between free radicals, inflammatory pathways and vitreoretinal proliferative disorders. It also confirms the pharmacological interest of prevention by antioxidants and free radical scavengers.

Inflammation measurement and immunocharacterization of cell proliferation in an experimental model of proliferative vitreoretinopathy.

An experimental model of proliferative vitreoretinopathy was developed in the rabbit eye by injecting a solution of human platelet-rich plasma. In this model we evaluated the progression with time of intraocular inflammation and the rate and origin of cell proliferation. A sterile solution adjusted to 107 platelets was injected into the right eye of a total of 46 pigmented and 14 albino rabbits. Animals were sequentially sacrificed at days 7, 14, 21 and 1 month after injection. Clinical evaluation of vitreoretinal proliferation, using a classification in six grades, and of anterior segment inflammation assessed by a Laser Flare Meter, were done for 1 month after injection, before histopathological analysis. Eighty percent of eyes developed tractional retinal detachment in 1 month. Histopathology showed intense cell migration and proliferation in the area of the ciliary body, as early as the seventh day, then further increasing rapidly. Infiltrates were composed of cytokeratin- and vimentin-expressing cells. Abnormal expression of vimentin was also found in ciliary and retinal epithelia and in M¿ller cells. Inflammation measured by the Laser Flare Meter was maximal at day 11 and then reached a plateau at significantly higher levels than controls. Albino rabbits showed significantly lower grades of proliferation, as compared to pigmented rabbits. This study thus clarified some characteristics of experimental vitreoretinal proliferations that that proved similar to those in human diseases, such as the involvement of ciliary body and retinal pigment epithelium, the existence of inflammatory reactions preceding cell proliferation and strong changes in intermediate filaments. This may provide a simple and valuable model for antiproliferative assays and shed some light on the pathogenesis of intraocular proliferative disorders.

Histopathological effects of topical ophthalmic preservatives on rat corneoconjunctival surface.

PURPOSE: Long term use of topical drugs has clearly been shown to induce toxic immunopathological changes in the ocular surface. However, little is known concerning the respective roles of active compounds and preservatives. Benzalkonium chloride (BAC) is the most used preservative and its cytotoxicity is well known, but other preservatives have not yet been clearly evaluated. We thus performed a comparative study to investigate toxic side effects induced in the rat ocular surface by applications of various preservatives, with special attention to inflammatory infiltrates. METHODS: A total of 35 brown Norway rats were divided into seven groups of five each. They received, for one month, in both eyes, either 0.01% cetrimonium chloride, 0.01% benzalkonium chloride, 0.01% benzododecinium bromide, 0.004% thiomersal, 0.05% methyl parahydroxybenzoate or phosphate-buffered saline (PBS), the last group remaining untreated. Then, animals were sacrificed and eyes were processed for histological and immunological procedures with monoclonal antibodies to rat immunocompetent cells. RESULTS: When compared to controls, all preservative-treated eyes consistently showed corneal and conjunctival damage, including epithelial alterations, various degrees of keratinization and inflammatory infiltrates at the limbus and within the conjunctival stroma and epithelium. No difference was found between the five tested drugs. CONCLUSIONS: This study confirms that most preservatives used in ophthalmic eyedrops may similarly induce strong histopathological and inflammatory changes in the ocular surface after short term use. Although obtained in animal model, these results confirm strong toxic side effects in patients with preexisting ocular surface disorders and/or receiving topical drugs for long periods.

Distribution of salicylate in pigmented rabbit ocular tissues after application of a prodrug, sodium monomethyl trisilanol orthohydroxybenzoate: in vivo and ex vivo studies.

SMB (sodium monomethyl trisilanol orthohydroxybenzoate) is an organic complex of silicium and salicylate and the main component of a collyrium used in lens transparency abnormalities. Biotransformation and penetration of salicylate in the whole eyeball have been investigated in vivo after repeated instillations of those 14C-radiolabeled eyedrops. We also studied more accurately the salicylate diffusion within the lens under ex vivo conditions. In vivo experiments demonstrated that 8 to 48 hours after the last instillation, radioactivity was detectable in most tissues and remained stable except in the chorioretina. The following gradient of distribution was observed: conjunctiva > cornea > iris-ciliary body > chorioretina > lens > vitreous body > aqueous humor >> plasma and blood. The diffusion of the radiolabeled compound in lens fibres was low, but a more important retention was observed in lens capsule. Though salicylate-metabolizing activities have been demonstrated in ocular tissues, no biotransformation could be detected under our experimental conditions. The lens SA-biotransformation activity was reported to be low and we can most probably consider that, in our ex vivo pharmacokinetic study, the lens metabolite amounts were negligible compared with the salicylate levels. Under such conditions, results showed that the salicylate reached a steady-state between 6 and 12 hours of incubation, characteristic of a passive diffusion mechanism. Quantitative image analysis of lens section autoradiograms revealed a more intense labeling of the anterior part of the lens and suggests that the lens epithelium may facilitate the salicylate diffusion. Furthermore, renal excretion is important since 40% of the administered eyedrops were eliminated during the study period.

Evaluation of antiproliferative effects of the somatostatin analogue somatuline in a rabbit model of traction retinal detachment.

As growth hormone (GH) and insulin-like growth factor type I (IGF-I) have been suggested to be involved in the development of some proliferative ocular disorders, we investigated the eventual antiproliferative properties of a long acting somatostatin analogue, somatuline or BIM23014 (IPSEN Biotech, France), in an original model of experimental proliferative vitreoretinopathy. Two studies were separately done to investigate respective effects of subcutaneously- and intravitreally administered somatuline. Injections of 10(7) human platelets freshly prepared from a unique normal donor were injected into the vitreous, cavity of pigmented rabbits. The first experiment consisted of evaluating vitreoretinal proliferation in 17 eyes from rabbits receiving subcutaneous injections of 25 micrograms/kg of BIM23014, given twice a day, from the day after injection for one month. A group of 14 eyes served as non treated controls. The second experiment was conducted in 33 eyes: 10 received intravitreally 1 microgram of somatuline given once a week for one month, 10 eyes similarly received 5 micrograms/week of somatuline, the remaining 13 eyes serving as controls with intravitreal injections of sterile saline. All animals were examined ophthalmoscopically twice a week for one month in a masked manner, and sacrificed at the end of the experiment for histological and immunohistological analyses. In all but two eyes from the subcutaneously treated group, intravitreal and preretinal membranes formed, five to eight days after platelet injection. Intravitreal proliferation progressively increased, resulting in various degrees of vitreoretinal retraction and retinal detachment.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of preretinal proliferation by free radical scavengers in an experimental model of tractional retinal detachment.

An original model of experimental proliferative vitreoretinopathy consisting of an intravitreal injection of 10(7) human platelets and 1 IU of hyaluronidase was developed in pigmented rabbits. One group of 11 eyes served as non-treated controls. Two other groups of 11 eyes each received Ginkgo Biloba extracts which are known free radical scavengers (EGb761, Ipsen, France), given orally in two doses, 50 mg kg-1 day-1 and 100 mg kg-1 day-1 respectively, from the day after the platelet injection to the end of the first month. The fourth group (11 eyes) was intravenously injected with a unique dose of 15000 U kg-1 of superoxide dismutase the day after platelet injection. All animals were ophthalmoscopically examined in a masked fashion twice a week for 1 month and killed at the end of the experiment for histological analysis. Vitreoretinal proliferation was graded according to a six-stage classification. The non-treated eyes showed a high rate of retinal detachment (11/11 eyes), with a mean final score of 3.91 +/- 0.94. Histologic examinations consistently showed retinal retraction by fibrocellular preretinal membranes spreading to both surfaces of the retina as well as preretinal neovascularization. Many cells positively reacted with anti-cytokeratin or anti-vimentin monoclonal antibodies. All three groups of treated eyes showed significantly lower scores of vitreoretinal proliferation at almost each time point of examination. At the end of the study, five retinal detachments were found in the EGb761 group at 50 mg kg-1 day-1 (mean final score 2.45 +/- 1.37), only one in the group receiving 100 mg kg-1 day-1 (mean score 1.64 +/- 1.03), and one in the SOD treated eyes. The lowest mean score found at day 28 was observed in the group receiving SOD (1.36 +/- 1.43), although this group presented during the first 3 weeks with an intense vitreous and sometimes anterior chamber inflammation. Statistical comparison between treatments did not show significant differences at most time points of the study. These results demonstrate that antioxidants may efficiently prevent preretinal proliferation, in clinicopathological entities where free radicals had not yet been shown to play a direct pathogenetic role. They are also among the first attempts for inhibiting preretinal proliferations with non-cytotoxic agents and using a non-ocular route.

CMDBS, functional analogue of heparin sulfate as a new class of corneal ulcer healing agents.

Soluble dextran polymer derivatives (CMDBSs) are originally synthesized as heparin-like plasma substitutes. Some of them mimic heparin in its interactions and stabilize, protect and facilitate actions of heparin binding growth factors. The wound healing activity of one specific CMDBS was studied in a model of corneal ulcer on the rabbit eye and compared with the activity of basic fibroblast growth factors (bFGF) added alone or in association with CMDBS. Total reepithelization was observed with bFGF + CMDBS, bFGF alone and CMDBS alone after, respectively, 3.8 +/- 0.78, 4.3 +/- 0.67 and 4.4 +/- 0.51 days. All treatments were efficient if compared with eyes treated with saline (p < 0.0001). The grade of significance of the applied treatments was as follows: bFGF + CMDBS > bFGF > CMDBS > saline. Our study pinpoints that some specific CMDBS are as potent agents as bFGF for corneal ulcer healing, and can therefore be proposed for therapeutic use.

Cytotoxicity studies in ophthalmology.

Ocular tissues and cells are more and more in direct contact not only with drugs but also with biomaterials, such as contact lenses, intraocular implants, corneal shields, and the cell reactivity study is an indispensable step before any clinical and human utilization. The cell toxicity may be direct, by cell membrane damaging, metabolic disturbance, or indirect by mitosis or cell differentiation blocking. In order to evaluate the unwanted effects, cell cultures are performed according to the drug or to the biomaterial to be tested: conjunctival and corneal epithelial cells, lens epithelium, ciliary processes epithelium... In this report, the cytotoxicity of three substances were evaluated on corneal cultured cells: Benzalkonium chloride (BAK), an ophthalmic preservative; Novesine (Oxybuprocaine + BAK), local anaesthetic; Neosynephrine (Phenylephrine chlorydrate), a commonly used mydriatic in ocular surgery. Results of cell counting (cell viability) are given according to curves and histograms (percentage of dead cells depending on time and doses). These data are discussed according to the different mechanisms of action of the three drugs BAK and Oxybuprocaine were found to exert a more direct cell toxicity whereas phenylephrine chloride acted indirectly by causing the sloughing of the cell monolayer.

Pharmacokinetic and autoradiographic studies of basic fibroblast growth factor on de-epithelialized and intact rabbit eye.

In a previous work, we showed and compared the wound healing properties of aFGF and bFGF topically administered on totally de-epithelialized rabbit corneas. Pharmacokinetic and autoradiographic studies were then performed to investigate the sites of accumulation of bFGF in ocular structures, both on de-epithelialized and intact rabbit eyes. After one single instillation of 125I-bFGF, all the ocular structures were dissected and the measurement of the radioactivity allowed to establish kinetic curves. The results showed a very important and early fixation of bFGF on denuded cornea (10 minutes) and a posterior distribution of the drug between 10 and 30 minutes. A second accumulation of bFGF in the anterior segment appeared 8 hours after application and then decreased till the 48th hour. These findings were confirmed by the macroautoradiographies and the microautoradiographies pointed out the fixation of bFGF not only at the location of the Bowman membrane, but also on the corneal endothelium. These experiments also demonstrated the systemic diffusion of bFGF into the untreated controlateral eye. The integrity of bFGF in the cornea and other structures was then confirmed by SDS PAGE followed by autoradiography. In the intact eye, bFGF was shown to penetrate in extremely low amounts, illustrating the major role of the corneal barrier. For a therapeutic use bFGF may be recommended as an efficient wound healing agent for epithelial but also endothelial defects. Its eventual unwanted side effects must be kept in mind to perfect an efficient low dose and short term clinical treatment.

Physiological effects of carbamylcholine, cyclic GMP and related agents on isolated rat ventricular myocytes.

The purpose of the present investigation was to ascertain the sensitivity of single dissociated myocardial cells to carbamylcholine and related substances. Heart cell cultures were obtained from newborn rat ventricles. The contractile activity was monitored by using a photoelectric transducer. The present results indicate that carbamylcholine alone or in association with sympathomimetic agents, i.e. isoproterenol, dibutyryl cyclic AMP and isobutylmethylxanthine, had no effect on the beating rate of the cultured ventricular cells. Moreover, dibutyryl cyclic GMP did not counteract the chronotropic response induced by either isoproterenol or dibutyryl cyclic AMP. Our findings failed thus to demonstrate the existence of activable muscarinic receptors in ventricular myocytes cultivated from the newborn rat and therefore supports the idea that the interaction between the adrenergic tone and the vagal innervation in the mammalian ventricle should be presynaptic. Furthermore, the present results seem to rule out the possibility of a mutual antagonism between cyclic AMP and cyclic GMP in this preparation.