Rona J. Delay

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 (∼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.

Role of Plasma Membrane Calcium ATPases in Calcium Clearance from Olfactory Sensory Neurons

Odorants cause Ca(2+) to rise in olfactory sensory neurons (OSNs) first within the ciliary compartment, then in the dendritic knob, and finally in the cell body. Ca(2+) not only excites but also produces negative feedback on the transduction pathway. To relieve this Ca(2+)-dependent adaptation, Ca(2+) must be cleared from the cilia and dendritic knob by mechanisms that are not well understood. This work focuses on the roles of plasma membrane calcium pumps (PMCAs) through the use of inhibitors and mice missing 1 of the 4 PMCA isoforms (PMCA2). We demonstrate a significant contribution of PMCAs in addition to contributions of the Na(+)/Ca(2+) exchanger and endoplasmic reticulum (ER) calcium pump to the rate of calcium clearance after OSN stimulation. PMCAs in neurons can shape the Ca(2+) signal. We discuss the contributions of the specific PMCA isoforms to the shape of the Ca(2+) transient that controls signaling and adaptation in OSNs.

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.

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.

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.

TRP Channels in Transduction for Responses to Odorants and Pheromones

Transient receptor potential cation channel, subfamily C, member 2 (TRPC2) is a TRP channel expressed by sensory neurons in the vomeronasal organ. Clearly the TRPC2 channel is critically important for transduction in the vomeronasal sensory neurons. However, it appears that TRPC2 is not the only channel that mediates chemical transduction in the VNO. In addition the transient receptor potential channel M5 (TRPM5) is expressed in approximately 5 % of the olfactory sensory neurons in the main olfactory epithelium where it plays a role in responding to intraspecific semiochemicals (chemicals involved in animal communication of the same species). Interestingly, TRPC2 is expressed in rodents but not in humans, whereas TRPM5 is expressed in the human raising the question for future work whether this TRP channel is involved in human semiochemical transduction.