Stanley A. Thayer

Regulation of calcium homeostasis in sensory neurons by bradykinin

The nonapeptide bradykinin (BK) activates sensory neurons and stimulates the transmission of nociceptive information into the CNS. We investigated the effect of this peptide on rat dorsal root ganglion neurons (DRG) grown in vitro. BK stimulated the synthesis of inositol trisphosphate (IP3) and the breakdown of phosphatidylinositol bisphosphate, the synthesis of diacylglycerol, and the release of arachidonic acid from DRG cells. The release of IP3 and arachidonic acid was not inhibited by pretreatment of the cells with pertussis toxin. BK also mobilized intracellular Ca2+ stores in DRG cells as assessed by fura-2-based microfluorimetry. Two types of Ca2+ stores appeared to exist in DRG neurons. One type could be mobilized by caffeine (10(-2) M), and this effect could be blocked by ryanodine in a use-dependent manner. These stores occurred primarily in the cell soma and were virtually absent from cell processes. A second type of store could be mobilized by BK, presumably through the mediation of IP3. These latter stores were distributed equally between the cell soma and processes. Experiments with combinations of caffeine and BK suggested that the stores mobilized by these 2 agents may be separate entities. Both the caffeine and BK sensitive Ca2+ storage sites appeared to participate in buffering a Ca2+ load induced in DRG neurons by cell depolarization. The relevance of these observations to the mechanism of action of BK on sensory neurons is discussed.

Measurement of neuronal Ca2+ transients using simultaneous microfluorimetry and electrophysiology

Fluorescent indicator molecules, such as fura-2, are useful probes for studying the concentrations of ions in single cells. A key feature of these fluorescent dyes is the shift in their excitation spectra upon binding the ion, thus making alternate excitation from two wavelengths desirable. In this report we describe an inexpensive system for making simultancous electrophysiological and dual excitation fluorescence measurements using equipment much of which is available in a typical biophysical laboratory. In order to synchronize the fluorescence signal with the appropriate excitation wavelength we devised a simple computer program which uses the output of photodiodes placed in the excitation beam to determine which wavelength is illuminating the cell. We also describe the use of a liquid light guide to transmit excitation illumination from the light source to the epifluorescence port of the microscope in order to isolate a perfusion chamber from light, electrical noise and vibration. A sensitive light detection system was assembled using a photomultiplier tube and discriminator that took advantage of sampling single unit events obtained with photon counting rather than the analog of annode current. However, rather than employing a sophisticated and expensive photon counting system, a filter was used to integrate the signal so that an analog output could be presented to a multichannel analog to digital converter commonly used for electrophysiological recordings. This apparatus was sensitive enough to allow the determination of the intracellular free Ca2+ concentration, [Ca2+]i, using fura-2 in the following situations: (a) in single processes of dorsal root ganglion (DRG) neurons grown in primary culture, (b) the release of Ca2+ from intracellular stores in single neurons, (c) the influx of Ca2+ through channels activated by excitatory amino acids and (d) it was also possible to measure [Ca2+]i transients while simultaneously recording Ca2+ currents at precisely controlled membrane potentials in DRG neurons. The instrumentation described here makes use of a number of innovations which investigators developing similar systems may find useful.

The effect of calcium channel antagonists on peripheral neurones.

The identification of high-affinity binding sites for dihydropyridines (DHPs) in the brain stimulated a search for effects of these and other organic Ca2+ antagonists on neuronal tissue.p2 Indeed, discovery of such effects could lead to novel therapeutic applications for these agents. Initially Ca2+ channel antagonists were not reported to have many effects on neurones and the question arose as to whether DHP binding sites in the nervous system really represented functional voltage-sensitive Ca2+ channels (VSCC). For example, DHPs were reported to be ineffective in blocking evoked neurotransmitter release suggesting that they did not block VSCC in nerve termin a l ~ . ~ . ~ However, it has now become abundantly clear that DHP-sensitive VSCC do exist both in the central and peripheral nervous systems.@ In addition to DHPsensitive VSCC, other types of CaZ+ channels also exist which are insensitive to DHPs and related agents.Owing to their accessibility, peripheral neurones have been important systems for examining the properties and functions of the multiple types of VSCC. We shall summarize these studies briefly here. Extensive reviews of VSCC in neurones can be found elsewhere?.

The Neuropharmacology of Ca2+ Channels

Ion channels that allow the influx of Ca2+ into neurons or other cells have been traditionally divided into two groups: voltage-sensitive Ca2+ channels (VSCC) and receptor-operated channels (ROC). Ca2+ influx via these and other pathways is one method by which an increase in the intracellular free Ca2+ concentration, [Ca2+]i, can be achieved. The second method is through release of Ca2+ from intracellular bound stores. Such increases in [Ca2+]i act as signals that trigger a large number of important neuronal responses. These include the release of neurotransmitters, the regulation of certain ion channels, modification of the cytoskeleton, and the production of long-term changes in the efficacy of synaptic transmission such as those associated with long-term potentiation (LTP). The division of ion channels into VSCC and ROC is somewhat artificial, however, as VSCC can certainly be modulated by receptor mediated events and some ROC can function in a voltage-dependent fashion under physiological conditions. In this chapter we shall focus on two examples. The first concerns the inhibitory modulation of VSCC in dorsal root ganglion (DRG) neurons by a variety of neurotransmitters such as neuropeptide Y (NPY). The second concerns the regulation of Ca2+ entry into central neurons by the excitatory amino acid neurotransmitter glutamate. This latter process involves the activation of a number of glutamate receptor subtypes and a number of ion channels.