Cyclophosphamide-Induced Inflammation of Taste Buds and Cytoprotection by Amifostine.

Taste buds in the oral cavity have a complex immune system regulating normal functions and inflammatory reactions. Cyclophosphamide (CYP), a chemotherapy drug, has wide-ranging disruptive effects on the taste system including loss of taste function, taste sensory cells, and capacity for taste cell renewal. In bladder epithelium, CYP also induces inflammation. To determine if CYP induces inflammation in taste buds, we used immunohistochemistry to examine tumor necrosis factor alpha (TNF-α) (a proinflammatory cytokine) expression over a 72-hour period. Expression of TNF-α increased in a subset of PLCß2 labeled (Type II) cells, but not SNAP-25 labeled (Type III) cells, between 8 and 24 h postinjection and declined slowly thereafter. This inflammatory response may play an important role in the disruptive effects of CYP on the taste system. Further, pretreatment with amifostine, a sulfhydryl drug known to protect normal tissues during chemo- or radiation therapy, reduced the amount of CYP-induced TNF-α expression in taste buds, suggesting this drug is capable of protecting normal cells of the taste system from adverse effects of CYP. Amifostine, used as a pretreatment to CYP and possibly other chemotherapy drugs, may offer clinical support for preventing negative side effects of chemotherapy on the taste system.

Transient Effects of Cyclophosphamide on Basal Cell Proliferation of Olfactory Epithelia.

Cancer is often treated with broad-spectrum cytotoxic drugs that not only eradicate cancerous cells but also have detrimental side effects. One of these side effects, disruption of the olfactory system, impedes a patient's ability to smell, perceive flavor, and ultimately may interfere with their nutritional intake and recovery from cancer. Recent studies reported that the chemotherapy drug, cyclophosphamide (CYP), can damage gustatory epithelia and disrupt cell proliferation in olfactory epithelia. In this study, we asked if CYP altered globose and horizontal basal cell proliferation in the murine main olfactory epithelium (MOE) and vomeronasal organ (VNO). We used antibodies for Ki67, a marker strictly associated with cell proliferation, and Keratin 5, a marker for the cytoskeleton of horizontal basal cells. Our results revealed a significant CYP-induced decrease in the number of proliferative cells in both epithelia, especially globose basal cells in the MOE, within the first 1-2 days postinjection. Recovery of cell renewal was apparent 6 days after injection. The immunohistochemical markers showed significantly higher levels of globose and horizontal basal cell proliferation in CYP-injected mice at 14 and 30 days postinjection compared with control mice. The prolonged proliferative activation of globose and horizontal basal cells suggests that, besides altering proliferation of olfactory epithelia, the epithelial substrate needed for successful cell renewal may be adversely affected by CYP.

Cyclophosphamide has Long-Term Effects on Proliferation in Olfactory Epithelia.

Chemotherapy patients often experience chemosensory changes during and after drug therapy. The chemotherapy drug, cyclophosphamide (CYP), has known cytotoxic effects on sensory and proliferating cells of the taste system. Like the taste system, cells in the olfactory epithelia undergo continuous renewal. Therefore, we asked if a single injection of 75 mg/kg CYP would affect cell proliferation in the anterior dorsomedial region of the main olfactory epithelium (MOE) and the vomeronasal organ (VNO) from 0 to 125 days after injection. Both epithelia showed a decrease in Ki67-labeled cells compared to controls at day 1 and no Ki67+ cells at day 2 postinjection. In the sensory layer of the MOE, cell proliferation began to recover 4 days after CYP injection and by 6 days, the rate of proliferation was significantly greater than controls. Ki67+ cells peaked 30 days postinjection, then declined to control levels at day 45. Similar temporal sequences of initial CYP-induced suppression of cell proliferation followed by elevated rates peaking 30-45 days postinjection were seen in the sustentacular layer of the MOE and all 3 areas (sensory, sustentacular, marginal) of the VNO. CYP affected proliferation in the sensory layer of the MOE more than the sustentacular layer and all 3 areas of the VNO. These findings suggest that chemotherapy involving CYP is capable of affecting cell renewal of the olfactory system and likely contributes to clinical loss of function during and after chemotherapy.

Cyclophosphamide and the taste system: Effects of dose fractionation and amifostine on taste cell renewal.

Chemotherapy often causes side effects that include disturbances in taste functions. Cyclophosphamide (CYP) is a chemotherapy drug that, after a single dose, elevates murine taste thresholds at times related to drug-induced losses of taste sensory cells and disruptions of proliferating cells that renew taste sensory cells. Pretreatment with amifostine can protect the taste system from many of these effects. This study compared the effects of a single dose (75 mg/kg) of CYP with effects generated by fractionated dosing of CYP (5 doses of 15 mg/kg), a dosing approach often used during chemotherapy, on the taste system of mice using immunohistochemistry. Dose fractionation prolonged the suppressive effects of CYP on cell proliferation responsible for renewal of taste sensory cells. Fractionation also reduced the total number of cells and the proportion of Type II cells within taste buds. The post-injection time of these losses coincided with the life span of Type I and II taste cells combined with lack of replacement cells. Fractionated dosing also decreased Type III cells more than a single dose, but loss of these cells may be due to factors related to the general health and/or cell renewal of taste buds rather than the life span of Type III cells. In general, pretreatment with amifostine appeared to protect taste cell renewal and the population of cells within taste buds from the cytotoxic effects of CYP with few observable adverse effects due to repeated administration. These findings may have important implications for patients undergoing chemotherapy.

Cellular mechanisms of cyclophosphamide-induced taste loss in mice.

Many commonly prescribed chemotherapy drugs such as cyclophosphamide (CYP) have adverse side effects including disruptions in taste which can result in loss of appetite, malnutrition, poorer recovery and reduced quality of life. Previous studies in mice found evidence that CYP has a two-phase disturbance in taste behavior: a disturbance immediately following drug administration and a second which emerges several days later. In this study, we examined the processes by which CYP disturbs the taste system by examining the effects of the drug on taste buds and cells responsible for taste cell renewal using immunohistochemical assays. Data reported here suggest CYP has direct cytotoxic effects on lingual epithelium immediately following administration, causing an early loss of taste sensory cells. Types II and III cells in fungiform taste buds appear to be more susceptible to this effect than circumvallate cells. In addition, CYP disrupts the population of rapidly dividing cells in the basal layer of taste epithelium responsible for taste cell renewal, manifesting a disturbance days later. The loss of these cells temporarily retards the system's capacity to replace Type II and Type III taste sensory cells that survived the cytotoxic effects of CYP and died at the end of their natural lifespan. The timing of an immediate, direct loss of taste cells and a delayed, indirect loss without replacement of taste sensory cells are broadly congruent with previously published behavioral data reporting two periods of elevated detection thresholds for umami and sucrose stimuli. These findings suggest that chemotherapeutic disturbances in the peripheral mechanisms of the taste system may cause dietary challenges at a time when the cancer patient has significant need for well balanced, high energy nutritional intake.

L-Amino Acids Elicit Diverse Response Patterns in Taste Sensory Cells: A Role for Multiple Receptors.

Umami, the fifth basic taste, is elicited by the L-amino acid, glutamate. A unique characteristic of umami taste is the response potentiation by 5' ribonucleotide monophosphates, which are also capable of eliciting an umami taste. Initial reports using human embryonic kidney (HEK) cells suggested that there is one broadly tuned receptor heterodimer, T1r1+T1r3, which detects L-glutamate and all other L-amino acids. However, there is growing evidence that multiple receptors detect glutamate in the oral cavity. While much is understood about glutamate transduction, the mechanisms for detecting the tastes of other L-amino acids are less well understood. We used calcium imaging of isolated taste sensory cells and taste cell clusters from the circumvallate and foliate papillae of C57BL/6J and T1r3 knockout mice to determine if other receptors might also be involved in detection of L-amino acids. Ratiometric imaging with Fura-2 was used to study calcium responses to monopotassium L-glutamate, L-serine, L-arginine, and L-glutamine, with and without inosine 5' monophosphate (IMP). The results of these experiments showed that the response patterns elicited by L-amino acids varied significantly across taste sensory cells. L-amino acids other than glutamate also elicited synergistic responses in a subset of taste sensory cells. Along with its role in synergism, IMP alone elicited a response in a large number of taste sensory cells. Our data indicate that synergistic and non-synergistic responses to L-amino acids and IMP are mediated by multiple receptors or possibly a receptor complex.

Identifying cellular mechanisms of zinc-induced relaxation in isolated cardiomyocytes.

We tested several molecular and cellular mechanisms of cardiomyocyte contraction-relaxation function that could account for the reduced systolic and enhanced diastolic function observed with exposure to extracellular Zn(2+). Contraction-relaxation function was monitored in isolated rat and mouse cardiomyocytes maintained at 37°C, stimulated at 2 or 6 Hz, and exposed to 32 µM Zn(2+) or vehicle. Intracellular Zn(2+) detected using FluoZin-3 rose to a concentration of ∼13 nM in 3-5 min. Peak sarcomere shortening was significantly reduced and diastolic sarcomere length was elongated after Zn(2+) exposure. Peak intracellular Ca(2+) detected by Fura-2FF was reduced after Zn(2+) exposure. However, the rate of cytosolic Ca(2+) decline reflecting sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA2a) activity and the rate of Na(+)/Ca(2+) exchanger activity evaluated by rapid Na(+)-induced Ca(2+) efflux were unchanged by Zn(2+) exposure. SR Ca(2+) load evaluated by rapid caffeine exposure was reduced by ∼50%, and L-type calcium channel inward current measured by whole cell patch clamp was reduced by ∼70% in cardiomyocytes exposed to Zn(2+). Furthermore, ryanodine receptor (RyR) S2808 and phospholamban (PLB) S16/T17 were markedly dephosphorylated after perfusing hearts with 50 µM Zn(2+). Maximum tension development and thin-filament Ca(2+) sensitivity in chemically skinned cardiac muscle strips were not affected by Zn(2+) exposure. These findings suggest that Zn(2+) suppresses cardiomyocyte systolic function and enhances relaxation function by lowering systolic and diastolic intracellular Ca(2+) concentrations due to a combination of competitive inhibition of Ca(2+) influx through the L-type calcium channel, reduction of SR Ca(2+) load resulting from phospholamban dephosphorylation, and lowered SR Ca(2+) leak via RyR dephosphorylation. The use of the low-Ca(2+)-affinity Fura-2FF likely prevented the detection of changes in diastolic Ca(2+) and SERCA2a function. Other strategies to detect diastolic Ca(2+) in the presence of Zn(2+) are essential for future work.

Odors activate dual pathways, a TRPC2 and a AA-dependent pathway, in mouse vomeronasal neurons.

Located at the anterior portion of the nose, the paired vomeronasal organs (VNO) detect odors and pheromones. In vomeronasal sensory neurons (VSNs) odor responses are mainly mediated by phospholipase C (PLC), stimulation of which elevates diacylglycerol (DAG). DAG activates a transient receptor potential channel (TRPC2) leading to cell depolarization. In this study, we used a natural stimulus, urine, to elicit odor responses in VSNs and found urine responses persisted in TRPC2(-/-) mice, suggesting the existence of a TRPC2-independent signal transduction pathway. Using perforated patch-clamp recordings on isolated VSNs from wild-type (WT) and TRPC2(-/-) mice, we found a PLC inhibitor blocked urine responses from all VSNs. Furthermore, urine responses were reduced by blocking DAG lipase, an enzyme that produces arachidonic acid (AA), in WT mice and abolished in TRPC2(-/-) mice. Consistently, direct stimulation with AA activated an inward current that was independent of TRPC2 channels but required bath Ca(2+) and was blocked by Cd(2+). With the use of inside-out patches from TRPC2(-/-) VSNs, we show that AA activated a channel that also required Ca(2+). Together, these data from WT and TRPC2(-/-) mice suggest that both DAG and its metabolite, AA, mediate excitatory odor responses in VSNs, by activating two types of channels, a TRPC2 and a separate Ca(2+)-permeable channel.

Calcium-activated chloride current amplifies the response to urine in mouse vomeronasal sensory neurons.

The vomeronasal organ (VNO) is an odor detection system that mediates many pheromone-sensitive behaviors. Vomeronasal sensory neurons (VSNs), located in the VNO, are the initial site of interaction with odors/pheromones. However, how an individual VSN transduces chemical signals into electrical signals is still unresolved. Here, we show that a Ca2+-activated Cl- current contributes approximately 80% of the response to urine in mouse VSNs. Using perforated patch clamp recordings with gramicidin, which leaves intracellular chloride undisrupted, we found that the urine-induced inward current (V(hold) = -80 mV) was decreased in the presence of chloride channel blockers. This was confirmed using whole cell recordings and altering extracellular chloride to shift the reversal potential. Further, the urine-induced currents were eliminated when both extracellular Ca2+ and Na+ were removed. Using inside-out patches from dendritic tips, we recorded Ca2+-activated Cl- channel activity. Several candidates for this Ca2+-activated Cl- channel were detected in VNO by reverse transcription-polymerase chain reaction. In addition, a chloride cotransporter, Na+-K+-2Cl- isoform 1, was detected and found to mediate much of the chloride accumulation in VSNs. Collectively, our data demonstrate that chloride acts as a major amplifier for signal transduction in mouse VSNs. This amplification would increase the responsiveness to pheromones or odorants.

Urine stimulation activates BK channels in mouse vomeronasal neurons.

Most odor responses in mouse vomeronasal neurons are mediated by the phospholipase C (PLC) pathway, activation of which elevates diacylglycerol (DAG). Lucas et al. showed that DAG activates transient receptor potential channels, subfamily C, member 2 (TRPC2), resulting in a depolarizing Ca2+ influx. DAG can be subsequently converted to arachidonic acid (AA) by a DAG lipase, the role of which remains largely unknown. In this study, we found that urine stimulation of vomeronasal neurons activated large-conductance Ca2+-activated K+ (BK) channels via AA production. Using isolated neurons, we demonstrated that repetitive applications of AA potentiated a K+ current that required a Ca2+ influx and was sensitive to specific BK blockers. Using immunocytochemistry, we found that BK channels are present in vomeronasal neurons with labeling on the soma and heavy labeling on the dendrite with a BK channel antibody. We examined the role of these BK channels in regulating neuronal firing when the neuron was activated by membrane depolarization or urine. Contrary to a recent report, our data suggest that BK channels contribute to adaptation of urine/odor responses because the inhibition of BK channels during urine stimulation promoted repetitive firing. These data strongly support the hypothesis that AA mediates an inhibitory pathway through BK channels, a possible mechanism for odor adaptation in vomeronasal neurons.

Gonadotropin-releasing hormone modulates voltage-activated sodium current and odor responses in Necturus maculosus olfactory sensory neurons.

The terminal nerve (nervus terminalis) extends from the basal forebrain to the nasal cavity and has been shown to contain gonadotropin-releasing hormone (GnRH). The specific function of the terminal nerve is unknown, but it has been hypothesized that it modulates the function of olfactory neurons. To examine the effects of GnRH on isolated Necturus maculosus olfactory sensory neurons (OSNs), we used the perforated configuration of the patch clamp technique to record current responses. GnRH had no effect on the membrane current at any holding potential but did modulate voltage-activated TTX-sensitive sodium current (INa). Within 1 min of applying GnRH, approximately 60% of the OSNs showed a decrease in the magnitude of INa. Initial responses to GnRH were inhibitory, although in one group of cells the initial inhibitory response was followed by a potentiation of INa with continual application (approximately 5 min). The time course of the GnRH response suggested that a second messenger pathway mediated the response. Inhibitors of PKC, tyrosine kinase, and PI3K were all able to inhibit the INa, but none of them could prevent the GnRH response. Application of a cAMP analog mimicked the effects of GnRH, and only inhibitors of PKA and PKG could prevent GnRH-induced inhibition of INa. This suggests that the modulation of voltage-activated sodium currents by GnRH involve a cyclic nucleotide pathway. In addition, GnRH modulated the odor responses of OSNs. Our data suggest the release of GnRH, presumably from the terminal nerve, can serve to modulate olfactory sensory neurons.

Pulse stimulation with odors or IBMX/forskolin potentiates responses in isolated olfactory neurons.

Many odor responses are mediated by the adenosine 3',5'-cyclic monophosphate (cAMP) pathway in which the cAMP-gated current is amplified by Ca2+-dependent Cl- current. In olfactory neurons, prolonged exposure to odors decreases the odor response and is an adaptive effect. Several studies suggest that odor adaptation is linked to elevated intracellular Ca2+. In the present study, using the perforated configuration of the patch clamp technique, we found that repetitive odor stimulation elicits a potentiation of the subsequent responses in olfactory neurons. This potentiation is mimicked by stimulating the cAMP pathway and does not appear to be related to phosphorylation of ion channels since protein kinase inhibitors could not block it. Our data suggest that local increases in [Ca2+]i via activation of the cAMP pathway mediate the pulse-elicited potentiation. In the first odor application, entry of Ca2+ through cyclic nucleotide-gated channels appears to be buffered. Repetitive stimulation allows local increases in [Ca2+]i, recruiting more Ca2+-dependent Cl- channels with each subsequent odor pulse.

Coupling between sensory neurons in the olfactory epithelium.

Coupling of olfactory sensory neurons (OSNs) in the olfactory epithelium of Necturus maculosus was demonstrated by dye-transfer with Lucifer yellow CH; however, the incidence of dye-transfer was low. Immunocytochemistry and Western blot analysis indicated that connexin 43, a gap junction channel subunit, was widely expressed by cells in the olfactory epithelium. Electrical coupling by presumptive gap junctions was assessed using electrophysiological recordings, heptanol block, tracer-uptake through hemi-junctions, and tracer-injection into tissue whole-mounts. Coupling, which involved pairs of OSNs only, was detected in approximately 3-10% of the OSN population; there was no evidence that OSNs were coupled into extended neural syncitia. These results suggest that coupling of OSNs by gap junctions is unlikely to have a general role in olfactory responses by mature (odor responsive) OSNs. Instead, the incidence of inter-neuronal coupling was small, similar to the fraction of immature OSNs, suggesting a possible role of gap junctions in the continual turnover and development of OSNs or possibly their senescence.

Two second messengers mediate amino acid responses in olfactory sensory neurons of the salamander, Necturus maculosus.

Odor transduction mediated by the adenylyl cyclase/cAMP pathway has been well studied, but it is still uncertain whether this pathway mediates the transduction of all odors in vertebrates. We isolated olfactory sensory neurons from the salamander Necturus maculosus and used calcium imaging with the indicator dye fura-2 to examine olfactory responses elicited by amino acids. The properties of approximately two-thirds of the odor responses suggested they were mediated by the adenylyl cyclase/cAMP pathway, but one-third of the responses were not mimicked by cAMP analogs nor blocked by inhibition of adenylyl cyclase, suggesting that these odor responses were mediated differently. Responses that were unaffected by inhibition of adenylyl cyclase were blocked by neomycin, an inhibitor of phospholipase C, implying that they were transduced by activation of phospholipase C. Some cells which responded to more than one amino acid appeared to employ both pathways, but each was used to transduce different odors. In addition, many responses that were mediated by the adenylyl cyclase/cAMP pathway were enhanced following inhibition of phospholipase C, suggesting that the phospholipase C pathway has a role not only in odor transduction, but also in the modulation of olfactory responses.